Test 4 Flashcards
What if the genetic information of a cell was protein and not DNA what observations would Avery and his team have seen?
The rough cells would not have transformed into smooth cells when the smooth cell free extract was treated with proteinase. The rough cells would have transformed into smooth cells when the smooth cell free extract was treated with DNAse whatever molecule allows the rough cells to transform into smooth cells is the bio molecule that contains the genetic information if you destroy that molecule, the genetic information or the “transforming molecule” is destroyed, and the rough cells do not have the instructions needed to produce the polysaccharide capsule and transform
What does this photo by Rosalind Franklin depict?
It is an x-ray defraction image by Roslyn Franklin that James Watson and Francis Creek used to deduce the structural properties of B form DNA
It does not determine the composition of DNA, which was studied by Chargaff
It does tell us the shape which is a helix. It tells us that DNA has a consistent diameter and it tells us that there are 10 base pairs per repeat.
What is the difference between negative and positive control in DNA transcription in prokaryotes?
We have to look at it from the point of view of transcription occurring based on the effect of a regulator molecule
In negative control transcription occurs until the regulator molecule stops it from happening ex lac operon
In positive control transcription occurs only when stimulated by a regulator molecule
True or false the repressor contains separate DNA binding domains and inducer binding domains
It’s true this is proved by the Jacob Minogue operon model where the super repressor will not interact with the inducer lactose which then causes it to permanently repress structural genes because the lactose is not able to allosterically regulate it and therefore it is able to bind to DNA
Alternative exon ‘exon skipping’
Skipping a spliceosome Rxn
Alternative 5’ splice site and 3’ sites
Just changing the 5’ slice site or 3’ splice site
Basically one part of the exon is removed vs the other which happens in different cells
What is the 5’ vs 3’ end
5’ means you have a free phosphate and 3’ means you have a free hydroxyl end
What is a unique linkage only found between the 5’methyl cap and the next nucleotide?
5’5’-triphosphate linkage
Also known is that not only is the guanine in the cap methylated but also are the 1st nucleotides after that cap
5’ methyl cap
The rna polymerase has enzymes needed for cap to attach to the 5’ end post transcription
What does tRNA do?
It carries an amino acid into the catalytic site of a ribosome, the tRNA base pairs to mRNA to ensure the selection of the correct amino acid for incorporation into a nascent polypeptide chain
Where do the RNA molecules come from?
The information for the RNA molecules are in our genome specifically within a gene
What is a gene?
It is a piece of DNA that includes something functional
It encodes an RNA molecule that will do its function in the cell, such as an RNA or a TRNA
It also can encode an mRNA that will be used to create a protein
Genes can also include any regulatory sequences that determine when that gene is expressed true or false
True
Where are the DNA sequences that determine when a gene is expressed?
They are also a part of the same gene that has the coding sequence for the RNA’s
They also determine when a gene is not expressed and what level it is expressed
What is another name for the RNA that is produced directly from the process of transcription
The primary transcript
- in prokaryotes the primary transcript, and the mRNA are the same exact thing because as soon as RNA is made from transcription, it is immediately used for translation with no processing in between
In eukaryotes, the primary transcript we make is the same as our mRNA true or false
False - the primary transcript which is then processed into its final form, the mRNA which will be used for the process of translation
What is the open frame or ORF?
It is usually referred to as the coding sequence which encodes the functional things like proteins
It is the portion of the gene that directly corresponds to whatever that functional thing is that it’s producing like a protein
What is a cistron?
It is another name for an open reading frame, which means it is a coding sequence that encodes a macromolecule
What is a gene?
The coding sequence and any regulatory elements that determine when that gene is used
What are the two factors that work together to regulate the levels of transcription?
The cis acting factors and the trans acting factors
What is a transacting factor?
It is a diffusible element, meaning it does not have a fixed place in the genome. It can go and affect transcription of one gene and then go to another gene and effect it’s transcription.
What are examples of transacting factors?
They are usually proteins, specifically, DNA binding proteins or transcription factors
What are transcription factors?
They are DNA binding proteins, which are also known as transacting factors that will scan the DNA and bind to a particular sequence and then they will affect transcription of the gene that sequence is associated with
What are the sites or DNA sequences that the transacting factors or DNA binding proteins bind to?
Those are cis acting factors
True or false usually when we are talking about this acting factors, we are talking about DNA sequences
True, there will be sequences located in a gene where one of the trans acting factors combine two which will either regulate the transcription or down regulate transcription depending on the transacting factor
What is going to work on jeans to determine when they are transcribed or when they are not transcribed?
Cis, and transacting factors
What is the enzyme that catalyzes transcription of DNA to RNA?
RNA polymerase
In what direction is transcription happening?
In the five prime to three prime direction just like DNA synthesis
When we are moving downstream towards the direction of transcription or towards the coding sequence, are the numbers getting larger or smaller?
The numbers are getting larger
What will RNA polymerase recognize in order to begin transcription?
It will recognize the promoter sequence, which is part of the gene, but not part of the coding sequence
What does RNA polymerase act like?
It acts like DNA polymerase
Therefore, aspartic acid, residues and magnesium ions are involved in transcription
What is a big difference between RNA polymers synthesis RNA versus DNA polymerase’s synthesizing DNA?
RNA polymer races synthesize RNA de novo meaning from scratch
What are the substrate for DNA polymerase?
Deoxy ribonucleoside tri phosphates
What are the substrates for RNA polymerases?
Ribonucleoside tri phosphates
-they will have 2’ hydroxyl groups in these molecules
What is a bigger deal making a mistake with RNA police or making a mistake with DNA polymerase?
Making a mistake with DNA polymerase because DNA is permanent in the cell so we will not degrade our entire genome or remake it because it is permanent. Therefore DNA polymerase is have proofreading from three prime to five prime via EXO nuclease activity which RNA polymerases do not have. 
What happens if a mistake is made while making RNA with RNA polymerase?
The RNA will be degraded or turned over in the cell and then we’ll use other RNA molecules for translation
True or false RNA polymerase is have exonuclease proofreading activity
False
During transcription, what is the strand that is going into the catalytic site?
The template strand, which is used as a template for RNA synthesis
The other strand in the DNA that we do not use for RNA synthesis is called the coding strand or the non-template strand
Why do we call the coding strand or non-template strand that name?
Because it’s the same sequence or same code as the RNA molecule that’s produced the difference being all the t’s are actually u’s in mrna
True or false the template strand, and the coding strand is the same for every single gene in the genome
False, the template strand, and the coding strand is defined differently for every single gene
What tells the RNA polymerase which strand to use in order to transcribe?
The promoter tells the RNA polymerase, which strand is the template strand in order to place that template strand into its catalytic site and remember that the promoter also tells the RNA polymerase in which direction it should be transcribing
What does holo enzyme mean?
It means core enzyme plus accessory subunits, which are additional subunits needed for the core enzyme to perform its job
What is an example of a holo enzyme in DNA replication?
DNA polymerase three
Holoenzyme in transcription is RNA polymerase which is made up of the core enzyme of 4 different subunits (2 alpha, beta, beta prime, and omega) as well as the accessory subunit, sigma
Where are the aspartic acid residues in our catalytic site of RNA polymerase coming from?
The beta and beta prime sub units because they form the catalytic site
What are alpha subunits important for in the RNA polymerase core enzyme?
There are important for enzyme assembly, and they will also interact with some transcription factors that
What do we believe the omega subunit of the core enzyme is involved in?
Stability
What is the holoenzyme a version of?
The polymerase that is going to start transcription ….. it will synthesize the first 10 nucleotides, then sigma leaves and the vast majority of RNA synthesis is done by just the core enzyme
What ability does sigma have that none of the other subunits have that are part of the Holoenzyme?
It has the ability to read DNA and find promoter sequences, which is important because promoters tell the polymerase the locations of genes
It is the only subunit that can locate the promoters …. without it, the polymerase cannot find where the beginning of a Gene is.
What is the most highly regulated step of transcription?
The initiation step which is also highly regulated in DNA replication
What is the elongation phase of transcription?
It is the synthesis of RNA in a five prime to three prime direction which is done by the core enzyme
What is the Terminator sequence?
It is a DNA sequence that is going to stop the process of transcription when RNA polymerase gets to that sequence
What are the three phases of prokaryotic transcription?
Initiation, elongation and termination
What is the main point of initiation in prokaryotic transcription?
To bring the holoenzyme to the promoter and start transcription
What is the RNA polymerase holoenzyme made up of?
The core enzyme and the accessory sub unit sigma
What is the job of the subunit Sigma?
To scan the DNA looking for DNA sequences, called promoters and binding to it, which tells the RNA polymerase that there is a gene it needs to transcribe
promoter indicates where transcription starts and in which direction it will go
What does the promoter tell RNA polymerase?
promoter indicates where transcription starts and in which direction it will go
What does the holo enzyme specifically recognize?
It looks for the -10 region sequence and the -35 region sequence of the promoter, which is upstream from the transcription start site indicated by +1
True or false what matters in the promoter region is the nucleotides in the -35 region and the nucleotides in the -10 region
False what matters is the spacing between the two regions which makes sure that both regions are positioned exactly where we need them
Why is the hollow enzyme instead of the core enzyme needed to find the promoter?
Because sigma is the only subunit that has the ability to read the DNA and find these promoters
How does sigma read the DNA and bind to the promoter regions?
It uses its amino acids to interact with the -10 and -35 regions and therefore reads the DNA and binds to it
What is the sequence that promoters have the highest affinity for?
The consensus sequence - it is drawn to the sequence and has many interactions with the sequence
A lot of prokaryotic promoters have sequences that are identical to the sequence or are nearly identical to it (similar)
What are the nucleotides particularly common at each position in the promoter which is part of the consensus sequence?
At -10, going from five prime to three prime we have TATAAT
At -35 going from five prime to three prime we have TTGACA
What is the additional element found in some prokaryotic genes but not in all of them?
The up element which is upstream from the promoter
It is A T rich - the alpha subunits of the holo enzyme are able to interact with it
What is the advantage that prokaryotes with the up element have over those who do not have it?
It gives more interactions between the DNA and the holo enzyme meaning these prokaryotes will carry out more transcription
Remember, the more interactions between the DNA and Holo enzyme means the more affinity the holo enzyme has for the DNA —-> the more RNA polymerase you will recruit——> the more transcription you will have
True or false the up element interacting with the alpha sub unit of the holo enzyme is enough to stimulate transcription
False, you will still need the interactions of sigma with the promoter region
What are constitutive promoters?
Many promoters and prokaryotes are constitutive
These are promoters that are always available for RNA polymerase to bind and stimulate transcription
What is another name for constitutive promoters?
Housekeeping genes - they are jeans that always need to be transcribed because they are the fundamental genes that the cell needs to do every day things like ATP Synthase. So it will always transcribe the ATP synthase gene and that gene will always have a constitutive promoter where RNA polymerase can always bind and activate transcription
True or false because a gene has a constitution for motor means it will always be transcribed at high levels
False
there are strong promoters meaning there is lots of transcription from that gene
And there are weak promoters Meaning you have low transcription from that gene
What determines if a constitutive promoter is a strong promoter or a weak promoter?
It is based on how close the particular promoter sequence is to the consensus sequence
Strong promoters have high sequence, similarity between the promoter and consensus sequence
Week promoters have low sequence, similarity between the promoter and consensus sequence
A mutation in a promoter that moves away from the consensus sequence decreases the rate of transcription initiation
What happens when the RNA polymerase is dealing with a strong promoter and what are the implications of that?
The implication is that the strong promoter has either an identical sequence to the consensus sequence or a very similar one which recruits the RNA polymerase very well and therefore results in lots of transcription
What is the implication of having an up element?
There will be an increase in transcription for genes that have an up element
It implies that particular gene that includes the up element has a stronger promoter than those that don’t because it is enabling more interactions between the holo enzyme and the promoter
What is the first step of transcription initiation?
The holo enzyme is going to come together or form from the core enzyme and sigma accessory subunit
Then it will start scanning DNA looking for promoter sequence
Once the hollow enzyme binds to the promoter, does it bind loosely or tightly?
It binds loosely - forming the closed complex
Why do we call the initial binding of the promoter with the holo enzyme the closed complex?
Because we have not yet separated the DNA strands yet
When will sigma be stimulated to open up the DNA during transcription?
Once there is a productive interaction between the hollow enzyme and the promoter …. It will break the hydrogen bonds between the nucleotides of each of the DNA strands which separates them
This allows us to put the template strand Into the RNA polymerase catalytic site
What is the open complex in transcription?
During transcription initiation, the open complex is when the DNA strands are separated by sigma of the Holo enzyme, resulting in a transcription bubble
When are we ready to start synthesizing RNA?
Once we form the transcription bubble
How many nucleotides will the hollow enzyme synthesize?
10 ~ 1 nucleotide per second (very slow)
Why is the initial rate of transcription or RNA synthesis slow?
Because there is tension or tug-of-war between the core enzyme that wants to move on from the promoter and Sigma, that wants to stay with the promoter it formed multiple bonds with after finding it
When will Sigma let go of the Holo enzyme?
Once it recognizes that transcription is productive because it already formed the first 10 nucleotides
When do we officially enter elongation?
When sigma breaks away from the core enzyme
What does the dissociation of sigma from the core enzyme allow the RNA polymerase to complete?
Promoter clearance which enables transcription elongation to begin
How fast is transcription during elongation?
About 50 to 90 bases per second
: there is a range because there are number of things that can affect the transcriptional rate of RNA polymerase
Things that can affect the rate is 1. torsional stress or super coiling happening ahead of the transcription bubble, which is relieved by topoisomerases (in DNA)
2. RNA secondary structures- complementary bases in the RNA can base pair and form hairpins and stem loops….
— tugging on the RNA which is still attached to the RNA polymerase catalytic site which slows down the polymerase
True or false translation can begin During transcription elongation in prokaryotes
True. Because there is no nucleus the translation machinery is near where transcription is happening therefore, translation machine machinery can grab onto the RNA that is coming out from the RNA polymerase that is still transcribing.
How many different types of termination are found in prokaryotic transcription?
Two rho, independent termination, and rho dependent termination
How is it determined for prokaryotes to use rho independent or rho dependent termination?
It depends on what sequences are encoded in the gene that is being transcribed
Rho independent termination is the mechanism used by the vast majority of prokaryotic genes for transcription termination
What is rho independent termination?
It is termination that happens without the protein, rho
Everything needed for this mechanism of termination is encoded in the gene itself
… meaning you do not need any additional proteins to terminate transcription
Two things are involved in this termination signal : 1. has sequences that will hydrogen bond and form a hairpin or stem loop structure (complementary sequences that form a secondary structure)
2. Immediately after the secondary structure forms, there will be a long repeat of U’s
RNA polymerase therefore transcribes the terminator sequences into RNA…. Those terminator sequences therefore form secondary structures on their own
Then the long repeat of U’s that are transcribed weakly base pair with the long repeat of A’s on the template strand (2 hydrogen bonds per pair) in the catalytic site
So, the secondary structures that forms tugs on the weak A U base pairs and tugs the RNA out of the catalytic site which ends transcription
What is involved in rho dependent termination?
This mechanism of transcription termination is dependent on a protein called rho
There is a different gene for rho, and that rho has to be produced itself
Somewhere in the gene is a sequence that encodes a binding site for this protein called rho…..it is called a rut element (rho utilization site)
—— this sequence will be transcribed into RNA by RNA Polymerase… and that sequence will become a binding site for the protein called rho
Rho recognizes the rut sequence and binds to it…. As a helicase it will use the power of ATP hydrolysis to travel up the RNA molecule in a 5’ to 3’ direction till it collides with the RNA polymerase that it caught up with (it rams into the polymerase) knocking the polymerase and RNA off which ends transcription
What is rho?
It is an RNA helicase —- it uses ATP to travel along RNA or DNA
RNA helicase will not bind to DNA
True or false prokaryotic genes will be continuously transcribed
False, that would be a huge waste of energy
It only makes the gene products when it actually needs the gene products
The cell is always about energy conservation
True or false eukaryotes also have operons
False only prokaryotes have operons
What are operons?
They are gene structures
that are clusters of coding sequences that are all transcriptionally regulated together instead of separately
Ex Operon for ATP Synthase which includes all the sequences, cistrons, or genes needed to create its subunits
——This prevents the prokaryotes from having to make a separate RNA molecule for every subunit of the ATP synthase
——- they can do this by organizing the atp synthase genes in a single operon that can be transcribed into a single mrna molecule which will contain all the different subunits of atp synthase ….. therefore every gene has its own ORF (open reading frame) in the operon coding for a particular ATP synthase subunit
Every operon therefore has one promoter and one terminator
(RNA polymerase will bind to the promoter and transcribe everyThing in the opera five prime to three prime until it reaches the terminator
sequence
What do operons do at the transcriptional level?
They allow all the genes or open reading frames within them to be regulated together
What happens to the sub units encoded in the open reading frames of the mRNA once they are transcribed from the DNA operon?
They are translated into proteins separately
What is a polycystronic mRNA?
It is the mRNA that encodes more than one macromolecule i.e. many different proteins from the cistrons of the DNA operon it was transcribed from ( that contains multiple open reading frames (multiple genes or sequences))
What is another word for open reading frame?
Cistron: a sequence that encodes a macromolecule i.e. a protein
For their genes, operons can have constitutive promoters what is an example of an operon that has a constitutive promoter?
ATP Synthase - prokaryotes always need ATP synthase to make ATP, so they will need constitutive promoters that are always available for RNA polymerase
Why are there alternatives to constitutive promoters?
Because not all genes are needed in all environments of the prokaryotic cells
Some of these genes are only needed in certain environments
What are inducible promoters?
These are promoters that can be turned on and off, depending on the environment “light switch”
Three things contribute to the level of gene expression from an inducible promoter. What are they?
- Specificity factors like sigma alter the specificity of RNA polymerase for a given promoter or set of promoters
- sigma regulates its specificity for a promoter
(meaning the interaction between sigma and the promoter is going to affect the rate of transcription from inducible promoters) - Repressors restrict access of RNA polymerase to the promoter
- Activators enhance RNA polymerase- promoter interaction
What are the sequences of DNA that the repressor will bind?
Operators which are downstream from the promoters
What are the activator binding sites?
They are DNA sequences upstream from the promoters where activators will bind to enhance the RNA polymerase-promoter interaction (up-regulate transcription)
In DNA, what are the larger regions exposing nitrogenous bases to the cellular environments?
Major Grooves
What are the smaller areas of DNA where less exposure of nitrogenous bases to the environment are occurring?
The minor grooves
How do you specificity factors like sigma or other proteins such as repressor or activators no what part of DNA to bind to to affect transcription
?
These proteins, use the major and minor grooves of DNA to interact and gain access to the nitrogenous bases which enables them to read the DNA sequences
Where does sequence specificity come from?
The nitrogenous bases not the sugar phosphate backbone which is the same throughout the DNA molecule
What are transcription factors?
The proteins that bind DNA and regulate transcription rates
Ex. Of trans acting elements
They are able to bind dna because they have a very specific structure in them called a dna binding domain (not all proteins have dna binding domains)
What is found in the DNA binding domain of transcription factors that is important for the transcription factors to be able to interact with DNA?
The recognition helix (special alpha helix)
-2ndary structure
-fits perfectly into the grooves of dna
-its amino acid side chains can reach into the grooves and allow it to interact (forming bonds) with the nitrogenous bases (sequences) which will allow the transcription factor to read the DNA (in order to find the right sequence)
——- the bonds the alpha helix amino acid side chains form with the nitrogenous bases are hydrogen bonds, and van der waals interactions
What makes sequence specific interactions with the DNA
?
The recognition alpha helix within the DNA binding domain of transcription factors
What stabilizes the sequence specific interactions of the recognition alpha helix with the nitrogenous bases in the grooves of DNA?
The amino acids elsewhere in the transcription factor protein that interact with the phosphate backbone of DNA
-they increase stability of the interaction
What should we remember for exposed base pair chemical groups?
For the major grooves of a T, no the pneumonic MADA : one methyl group (on T), one H-bond acceptor (T), one H-bond acceptor (A), one H-bond donor (A)
For the major grooves of GC : Remember HADA - Other H (C) , H-bond donor (C), 2 H-bond acceptors (G)
For the minor grooves exposed base pairs?
AT- each base has a h-bond acceptor, but purine (A) has other H
Same for TA
GC- each base has h-bond acceptors, but purine (G) has the h-bond donor
True or false transcription factors can tell all four base pairs apart from each other using major groove interactions
True - Can distinguish between AT, TA, CG, and GC pairs
High sequence specificity
What do transcription factors distinguish in the minor grooves and does it have as much sequence specificity as in the major grooves?
They distinguish (AT/TA) from (GC/
CG)
Less sequence specificity than the major grooves where we distinguish AT from TA from GC and CG
Why do the vast majority of transcription factors interact with the major grooves of DNA?
Because the major grooves contain more sequence specificity than the minor grooves
Which transcription factor interacts with the minor groove and why?
Sigma interacts with the minor groove of the promoter because it is able to distinguish between Slight variations in the promoter sequence, and still bind to it
What are the most common amino acids used to bind DNA and is there a clear code for which base pair is recognized by an amino acid?
Asn, Gln, Glu, Lys, Arg
There is no clear code : it depends on the aa’s position in the alpha helix and what amino acids are next to them.
What is the most common structure that makes up DNA binding domains of transcription factors?
The helix turn helix motif
Both helices are alpha, but the 2nd mentioned is the recognition helix
The 1st doesnt interact with DNA - it is primarily there for structural purposes
What do you transcription factors almost always bind as?
As dimers or higher order ligamer
Dimer means the protein is made of two subunits: two of the same makes a homodimer, and two different ones make a heterodimer
Each subunit will have a helix turn helix motif (so each will have a recognition helix)
In order for transcription factor to make productive interactions with DNA every helix turn helix motif in the subunits regardless of the ligamer order must interact with their particular sequence
Why is it important that we have a dimer trimer or Tetramer in prokaryotes?
Overall, you are increasing the specificity of the reaction based on how many subunits you have in the DNA Binding Domain of the transcription factor
Because multiple subunits increases necessary sequence specificity (each subunit recognizes its particular sequence) and those sequences in the DNA have to be positioned perfectly a part from each other in order for the protein to bind (ensuring the protein only binds to very specific sites in the DNA
Besides increasing the specificity of the protein, what else does having multiple subunits in a transcription factor accomplish?
Stability - there will be more interactions with the sequence whether there are more H bonds or van der waals interactions with the bases, or another part of the subunit is forming electrostatic interactions with the phosphate backbone of the DNA
What is negative gene regulation?
It means we are negatively affecting transcription further means that we are pressing transcription
We understand that transcription factors have to bind to DNA in order to confirm the sequence they will be affecting, but how do they actually activate or repress transcription?
Through positive and negative gene regulation
In the relationship of a protein binding to a DNA specific sequence, and negative repression, which is the transacting element and which is the cis acting element?
The cis acting element is the operator (DNA specific sequence), and the transacting element is the repressor (transcription factor)
True or false the operator is never embedded in the promoter?
False sometimes the operator is embedded in the promoter, but many times it is downstream from the promoter region
But it is always in between the promoter and the transcription start site at +1
How do repressors know when to turn off gene expression and when to allow it to be on to allow transcription to occur?
Compressors are going to respond directly to the environment that the cell is in
- small molecules in the environment will change its activity. They are called effector molecules.
- effector molecules relate information about the environment to the transcription factor
How do effective molecules relate information about the environment to the transcription factor?
They bind the transcription factor and they change its shape
What are the two ways that repressor’s can be negatively regulated?
A. Oppressors will bind to the DNA in the absence of the effector molecule. When the effector molecule is present in the scenario, it will find to the repressor and hide it recognition helix which will prevent it from binding to the operator or DNA specific sequence.
B. The repressor will only bind to the operator in the presence of the effector molecule which will expose its recognition helix. Therefore, in the absence of the effector molecule, the repressor recognition helix remains hidden, and therefore it is unable to bind to the operator or DNA specific sequence.
What happens in positive gene regulation?
You are promoting transcription using positive gene regulation
The stimulation of transcription using positive gene regulation is going to be done by proteins called activators that enhance the RNA polymerase - promoter interaction
In the relationship of transacting to cis acting binding what is the acting element and transacting element in positive gene regulation?
The cis acting element is the activator binding site
The trans acting element is the activator
How do you activators stimulate transcription? Where are they located?
The activator binding site is upstream from the promoter site
So not only is the holo enzyme bound to the promoter through sigma interactions, but the activator is bound to the activator binding sites which then has interactions with the alpha subunits of RNA polymerase
Therefore, there are more interactions that are a polymerase will have with the promoter proximal region, which means RNA polymerase has a higher affinity for that promoter and subsequently, more RNA polymerase will be recruited to get more transcription initiation
What is an implication of the activators responding directly to the environment?
There will be two different mechanisms for positive gene regulation based on the presence or absence of an effector molecule
A. So in this mechanism, the activator will only bind to it activator binding site in the presence of an effector molecule. And it’s absence it will not bind to the activator binding site.
B. In this mechanism, the activator will only bind to the activator binding site in the absence of the effector molecule. In its presence, it will not bind to the activator binding site.
What does the lac operon encode?
Multiple enzymes needed to break down lactose to make energy
It is used when glucose is absent, and the cell needs a secondary carbon source so it will activate transcription of the lac operon , making enzymes needed to produce energy from the lactose
Why is glucose equalize main source of energy or its favorite food?
Because E. coli does not have to use a lot of energy to break down the glucose and it gets its most amount of energy by breaking down glucose
True or false the lac operon has a constitutive promoter
False the lack opera has an inducible promoter that can be turned on and off, based on the presence or absence of lactose
If there is no lactose in the environment, the lac operon’s transcription will be repressed to save energy by not making the enzymes needed to break down lactose
What kind of ligamer is the lac repressor?
This transcription factor is a homo tetramer, whose subunits each have a helix turn helix motif to bind it to a very particular sequence in the operator (make productive interactions with the DNA)
Where are the operator sites in the lac operon?
The main operator site O1 is found downstream from the promoter but upstream from the plus one transcription start site
02 is within the coding sequence
03 is upstream from the promoter
These are the sites that the lac repressor will interact with
Which operator site does the lack opera always have to interact with?
O1 - one of the dimers of the lac repressor will always bind here for the repressor to work
The other dimer region will either act with O2 or 03 it is random
What is the effector molecule in the lack opera and what kind of regulation is it employing?
Allolactose is the effector molecule which is present when lactose is present
It uses negative gene regulation to bind to the repressor, hiding its recognition helices, so ultimately keeping it from binding to the DNA specific sequences (operator regions)
Why is cyclic AMP the effector molecule for our activator of the lac operon?
Because it is produced by adenylyl cyclase when there are low levels of glucose
So it stimulates the transcription of operons that are able to break down secondary carbon sources such as the lac operon
Which activator is camp the effector molecule for?
Camp receptor protein or CRP
- it is a homodimer and each of its two subunits is where the cyclic AMP binds
It is employing the positive gene regulation where the effector molecule cAMP has to be present in order for the activator CRP to work
- therefore if we have high glucose levels and therefore low cyclic AMP (effector molecule), the activator CRP will not bind, and therefore transcription of the lac operon will not be stimulated
However, when glucose levels are low and cyclic, AMP levels are high then the cyclic AMP (effector molecule ) will bind to the CRP (activator) and change its shape by exposing its recognition helices so that it combines with the DNA
How is CRP stimulating transcription?
It will interact with the alpha subunits of the RNA polymerase holo enzyme through amino acids which are part of its structure
- therefore it directly interacts with the holo enzyme which increases the holoenzyme’s affinity for the promoter, and also increases how much polymerase will get to the promoter, and therefore how much transcription will result
In scenarios where lactose is absent, and therefore the amount of glucose does not matter in when considering the transcription of the lac operon is there ever no transcription and why?
No, there will always be a very very low amount of transcription because there is an equilibrium between the DNA specific sequence and the transcription factor which in this case is the repressor with the DNA/repressor complex because of the very weak non-covalent interactions between the DNA specific sequence and the repressor Which means that there will be small amounts of the repressor that dissociates from the DNA, which allows a very, very low level of transcription
Is the promoter of the lac operon weak or strong and what makes it weak or strong?
The lac operon promoter is weak because it’s sequence deviate from the consensus sequence, which is also why an activator is necessary in order for it to have a high level of transcription
Is the binding of the repressor with the effector molecule in the lac operon the reason for transcription or gene expression happening?
No, in the presence of high amounts of glucose, it is the weak promoter’s interaction with the sigma subunit of the holo enzyme that allows transcription or gene expression of the lac operon to happen
- the repressor being bound by allolactose, which is the effector molecule, for it, just allows the lac operon to be on or functional so that when the sigma sub unit binds to the promoter, then gene expression or a transcription can happen
Since eukaryotes do not have operas like prokaryotes, what are our gene structures?
They are standalone transcriptional units
This means we do not have operons that have multiple open reading frames for different proteins in the same gene structure
True or false eukaryotic genes have one promoter and one terminator like prokaryotes
False eukaryotic genes can have multiple promoters and multiple terminators
True or false the coding sequence or open reading frames of eukaryotes are going to be interrupted in the DNA by non-coding sequences
True. The non-coding protein sequences are called Intron that interrupt our open reading frames which include exons which are protein coding sequences
True or false RNA pries does not know the difference between exons and introns
True the polymerase will bind to the promoter region of a gene, and it will transcribe everything from the transcription start site all the way down to the terminator site sequence in a five prime to three prime direction continuously
Why are the primary transcript and the final mRNA two different things in eukaryotes?
Because the primary transcript transcribed from the DNA sequence of the gene by RNA polymerase contains intron or non-coding sequences in between axons or coding sequences that need to be spliced out or removed to result in our final mRNA that will be used for translation
True or false epigenetic regulation of transcription happens in both prokaryotes and eukaryotes
False, genetic regulation, only happens in eukaryotes, which has to do with our genome structure and the way we package it in our cells
True or false all of our cells, regardless of their differences in phenotype/function have the same genome
True.
This is possible because no one cell in our body is going to express all 29,000 different jeans that are part of the Genome that each cell has
Instead, each cell is going to express a population of those jeans to give it a cell specific expression
How do ourselves express only a population of the genome each cell has to express specific expressions or phenotypes?
Regulation of the gene expression at cell specific levels is what results in our different cell types
Is transcription the only place where gene regulation of expressed protein in a cell happens ?
No overall expression of functional proteins are regulated at the level of :
Transcription (primary level) - it is the thing that most affects overall gene expression
RNA Processing
MRNA turnover
Translation
Post-translational modification
Cellular Trafficking
Protein Turnover
“PCP TRMT” - PCP Treatment
The majority of the cell cycle our chromosomes are condensed true or false?
False our chromosomes are condensed during mitosis, which helps us to separate DNA into different daughter cells. However, our chromosomes are not condensed during interphase, which is every other phase but mitosis in the cell cycle (G1, S, and G2)
So in every other part of the cell cycle, but mitosis genome is going to be a lot more diffuse (allows us to access the dna easier)
What is chromatin made up of? And what does it make up?
DNA proteins and RNA
Chromatin makes up chromosomes
Think scarf (chromosome), and the scarfs threads (chromatin)
This structure is very dynamic and it will change throughout the cell cycle
What is chromatin remodeling?
Structural changes in the chromatin
Two structures of chromatin found in the nucleus through most of the cell cycle, which is interphase:
Heterochromatin- very condensed because there are lots of proteins involved where the DNA is very tightly wound around those proteins
Therefore, the DNA is not accessible in these structures
Therefore, these structures are transcriptionally silent areas of the genome
Ex of a structure always in the heterochromatin state is the center mirror which do not have jeans that need to be transcribed so the DNA is kept tightly wound around its proteins which also keeps it organized
Ex 2: telomeres is another example of an area of our chromosomes that is always in a heterochromatin state: there are no genes here
Ex. 3 : cell type specific genes may be in a heterochromatin state in certain cells because they are never used in those cells
Euchromatin- these structures have less proteins than there are in heterochromatin structures …. it is a less condensed structure…. so DNA is more easily accessible…. therefore, we see active transcription of jeans and chromatin that is in a euchromatin state.
How is the chromatin structure going to contribute to the regulation of transcription?
What is the basic unit of chromatin?
Nucleosome
“ individual fibers (nucleosomes), make up the thread (chromatin) of the scarf (chromosomes)”
- They have 8 histone proteins that make up the histone core : 2 copies of 4 proteins - H2A, H2B, H3, H4
- 200 base pairs of DNA : 147 wraps around histone core 2x , and the other 50 bps are linker DnA between nucleosomes
What is the 10 nm fiber?
It is the diameter of a nucleosome
And nucleosomes are beads on a string, which is the structure in euchromatin
Whenever a gene needs to be actively transcribed, what is the first thing that the cell has to do?
It has to put its chromatin structure into the beads on a string structure so that the RNA polymerase and transcription factors can gain access to the DNA and actually transcribe the gene
True or false his stone proteins are very positively charged proteins
Do they make sequence independent interactions with DNA?
True
Yes they make sequence. Independent interactions with DNA because we want our entire genome to be coded in these histone proteins which allows them to bind anywhere in the genome
Therefore, they interact with the negatively charged sugar phosphate backbone
- this makes sense because the sugar phosphate backbone is the same throughout the entire DNA molecule
—— there is no sequence specificity in the sugar phosphate backbone
So what are the interactions between the positively charged amino acids of the histone proteins and the negatively charged sugar phosphate backbone, which allows DNA to wrap around the histone protein?
Electrostatic interactions allowed the very flexible DNA molecule to wind around the histone core
What does the histone protein H1 do and is it a part of the nucleosome?
Although it is not a part of the his stone core, it is an additional histone protein that may be present at a nucleosome
H1 binds to the linker DNA connecting the two nucleosome to each other, which prevents the DNA from falling off the histone core
What is the importance of the histone proteins having disordered or undefined structural tails?
Those tails that reach out from the his stone core have amino acids that interact with the DNA of the neighboring nucleosome, which means they can interact with the amino acids of the neighboring histone core protein tails
Our cells can modify these interactions to either encourage more interaction meaning they will bind tighter together and form a condensed structure
or
the cells will cause these histone tails to interact less with each other, making them more spread apart and therefore more open
What kind of modifications can our cells make to change the interactions between the nucleosomes? Are they covalent or non covalent?
We use epigenetic markers or epigenetic modifications, which are covalent modifications to regulate transcription via our DNA or to these nucleosomes
- therefore we are not changing our genetics because the level of regulation is epigenetic or above genetics (we are not changing the dna sequence)
—— we are changing the chroma structure and how transcription machinery interacts with the DNA to mediate transcription. This is a genetic or above genetics.
True or false epigenetics like genetics can be changed by your environment
False epigenetics can be changed by your environment, but genetics cannot be changed by your environment
What are the Histone tails and where are they located?
They are N termini of histones H3 (1 tail per protein) and H4 (1 tail per protein)
They are N and C termini of H2A (2 tails per protein: one from N terminus end, and 1 from the C terminus end) and H2B (2 tails per protein)
Each tail has amino acids that we can modify to mediate the interactions between the nucleosome
What are two modifications of histone tails?
- Acetylation / De-Acetylation of histone tails causes the chromatin to open or close
——histone acetyltransferase (HAT) acetylates lysine in the histone tails
——histone deacetylase (HDAC) removes acetyl groups from the histone tails
Remember that histone is a very positively charged protein because of these lysines which are positively charged (acetyl groups are negatively charged) — their charges will be neutralized which enables the opening of the chromatin structures, making the DNA more accessible
-so acetylation is associated with chromatin, and therefore actively transcribed genes
-Remember that the acetyl groups come from acetyl CoA
-so when a Gene no longer needs to be transcribed, we will close up the structure by removing acetyl groups, which will bring nucleosome closer to each other and condense the euchromatin structure (it is still euchromatin because there are not as many proteins in this structure as there are in heterochromatin structures)
True or false the interactions between HATs and HDACs are reversible reactions in euchromatin
True
Is methylation as easy to reverse as acetylation once you do it?
No
Is acetylation or methylation found in both euchromatin and heterochromatin
?
Methylation
Hypermethylation of histone proteins are typically associated with a heterochromatin state which means you’ll see a lot more methylation in Heterochromatin (transcriptionally silent state) than in euchromatin
What enzyme will methylate euchromatin or heterochromatin?
Histone methyl transferase (HMT)
It will transfer metal groups onto lysine or arginines of the histone tails
Why does acceleration of his stone tails lead to the opening of euchromatin?
Because prior to ace ation, the lysine of the histone tales are able to electrostatically interact with neighboring DNA on other nucleosomes via its positive charge, however, once acetylated, the lysine becomes neutralized and loses its ability to electrostatically interact with the DNA of the other nucleosomes which causes loosening and opening to the beads on a string structure of euchromatin
This is due to less interactions between the nucleosome overall, which then allows more accessibility for things like transcription machinery
What else does acetylation do to enable transcription?
When lysines are acetylated, they become a binding site for chromatin remodeling complexes, which are also instrumental in making the euchromatin transcriptionally active
What effect methylation has depends on what?
- specifically what amino acid of the histone is being methylated (lysine or arginine)
- how many methyl groups are on that amino acid (mono, di, or trimethylation)
Typically, we see hypermethylation which is typically associated with trimethylationl
Why do we typically see hyper methylation or tri methylation and heterochromatin structures?
Because the trimethyl group becomes a binding site for proteins that are involved in condensation of the chromatin which forms the heterochromatin structure
How do mono methylation and dimethylation differ from trimethylation’s?
They are binding sites for things that are needed in transcription
We see them in actively transcribed genes because certain transcription factors or other things recognize those sites as binding sites
What is the real difference between euchromatin and heterochromatin?
Euchromatin has a lot of diversity with its epigenetic markers during its transcriptionally active state (acetylation, mono, di, and trimethylation) than the very similar markers in heterochromatin (lots of trimethylation)
What is Epigenetic modifications?
Regulation of gene expression via covalent modifications that cells make to our chromatin that does not change the genetics but changes the accessibility of the DNA to transcription factors ( proteins needed for transcription)
What are epigenetic changes that we can make to the DNA sequence and does it change the genetics?
Epigenetic changes to the DNA sequence is not changing the base pairing between the strands of DNA, but we are making changes that will change the accessibility of the DNA to our transcription machinery
What is different when we methylate our DNA then when prokaryotes methyl theirs?
Our DNA is not methylated throughout our entire genome. It is much more targeted specific methylation that we are doing, which affects gene expression.
Where is our DNA methylated?
Wherever there is a CG (CpG) sequence
DNA methyltransferase (DNMTs) which are different than HMTs (catalyze methylation of proteins) for histones, catalyzes the methylation of DNA sequences (CG) more specifically the C of the CG sequence (will be methylated on the 5 position making 5 methyl cytosine)
Where is methylation of the CGs going to take place?
On sequences with many CG pairs called CpG islands which are sequences that are promoter proximal (near the promoters)
The methyl group on the C of the CG pair is in the major groove which is bulky and prevents transcription factors, specifically activators from binding to the DNA , which prevents them from stimulating transcription
So, the trend is that when CpG islands are methylated then that particular gene is transcriptionally silent
The exception to the rule are activators that only bind to CGs (this is minority of the time) because majority of the time hypermethylation of CpG islands is associated with transcriptional silencing since transcriptional activators can no longer bind to those sites
Regulation of transcription is regulated by interactions between what?
Between cis and transaction factors
True or false our regulation of gene transcription needs activation, which is different than prokaryotes
True. The vast majority of eukaryotic gene expression regulation is positive regulation . Meaning we have to do things in order to stimulate transcription.
—-The default state of our genes is to be off and we only activate them when we need those gene products.
In prokaryotes, their default state is on and they have their DNA exposed to be able to bind the RNA polymerase and then when they don’t need it, they just repress the transcription
What is the difference between our RNA polymerase and the RNA polymerase in prokaryotic transcription?
Our RNA polymerase is not able to find promoters on its own (we do not have a sigma sub unit)
This is why we have special transcription factors which we call general transcription factors that bring RNA polymerase to the promoter in order to begin transcription
What are the cis acting elements in eukaryotic genes?
The Tata box is our promoter
Promoter proximal elements - We also have an activator binding site which are promoter proximal Just like in prokaryotes
Enhancers - they are promoter distal sites, which are far away from the promoters (very far away but can still have an effect on the promoter’s transcription initiation)
——-they are clusters of finding sites for transcription factors
I.e. One enhancer is going to have multiple sequences in it for multiple different transcription factors…… so one enhancer can have multiple transcription factors bound to it
Architectural Regulators
Which cis element dictates to RNA polymerase which promoter to use?
Enhancers
True or false enhancers can be upstream or downstream from promoters
True enhancer’s can be both upstream and downstream from promoters, and of course, distal that is far away from the promoters
Can enhancers be found in Intron of the coding sequences?
Yes, they can and therefore transcription factors can find their in order to stimulate transcription
Since we have more than one promoter in eukaryotes, how does the RNA polymerase know which promoter to use?
Which promoter is used depends on which enhancers are used, which depends on what specific transcription factors a specific cell uses
Ex .
The transcription factor specific to one cell may bind to either upstream enhancers, which tells them to bind to promoter one versus transcription factors specific to another cell that can bind to enhancers downstream, which will tell them to bind to promoter two
How do enhancers which are very distal to promoters able to affect transcription initiation at the promoter?
Architectural regulators are proteins in eukaryotic cells that take advantage of DNA‘s flexibility by binding to the DNA and changing its shape and bringing the enhancer region, physically closer in proximity to the promoter, which is necessary because now the activators at that enhancer site (promoter distal sites) are physically in close proximity to the RNA polymerase complex at the promoter and can interact with it helping to stimulate transcription
What is between activators and RNA polymerase’s that mediate their interaction?
CoActivators
They do not find to DNA
Because they do not have DNA binding domains
Instead, they are making protein, protein interactions, mediating the interactions between the activators and RNA polymerase by binding to the activator and to RNA polymerase
What do transcription factors have that enable them to bind to DNA? This is found in both eukaryotes and prokaryotes.
They have a DNA binding domain, which is a specific structure within their subunits that contains at least one alpha helix, and that alpha helix is going to stick most likely into the major groove of DNA making sequence specific interactions with the DNA remember that this alpha helix is called the recognition helix
What motif do we share with prokaryotes in regard to specific DNA binding domains in our transcription factors?
Helix turn helix is the structure that can make up that DNA binding domain in both prokaryotes and eukaryotes
What other structures can exist in our DNA binding domains of the transcription factors that makes them more variable than found in prokaryotes?
We have zinc finger transcription factors which have zinc finger motifs in the DNA binding domain
They are made up of 30 amino acids
They also have anti-parallel, beta sheets at the N terminus followed by a turn, and then an alpha helix, which is the recognition helix,
which will stick into the major groove of DNA making the sequence specific interactions with DNA
(the amino acids on the recognition helix have side chains that will stick into the groove of DNA to facilitate the sequence specific interactions)
The two histidine’s on the C terminal side and the two cysteines on the N terminal side do not interact with DNA at all their purpose is to position that zinc ion to give the structure its overall three dimensional shape.
Monomer can have multiples of these zinc, finger structures that can increase stability and specificity just as the higher order ligamers in prokaryotes
How do we not lose stability and specificity of a reaction when we use a monomer as opposed to higher order ligamers definitely used by prokaryotes?
The one monomer has multiple DNA binding domains within it, which users their recognition helices to interact with different portions of the DNA in major grooves
True or false eukaryotes also use higher ligamers as transcription factors like prokaryotes?
True
an example is the leucine zipper motif
And helix turn helix
What makes the leucine zipper motif different than the helix turn helix and zinc finger motifs?
It has two jobs
- it binds DNA and a sequence specific manner
- it dimerizes two subunits together, so the interactions between the individual subunits will also happen in this DNA binding domain
It has two alpha helices that come together to form a leucine zipper structure ( both have many leucines that interact with hydrophobic amino acids on the other alpha helix dimerizing them or bringing the two subunits together )
The extension of these alpha helices have the amino acids that are making sequence specific interactions with DNA
How many regulatory sites in eukaryotic genes have to be activated by transcription factors in order for you to get transcription in eukaryotes?
Six or more regulatory sites
How do we get cell specific expression that makes each cell unique (which makes cells look and function differently ) when we have 29,000 different genes in every cell with only 3000 transcription factors in every cell?
Think of a closet with 30 shirts 30 pairs of pants and 30 shoes
That does not mean we have 30 outfits. That means we have a lot of outfits because we can mix and match the shirt and shoes to make a plethora of different outfit combinations.
It is the same with our
Transcription factors no one of the cells is going to express all 3000 transcription factors each cell is going to express a population of those transcription factors
Ex.
Kidney cells express a certain population of transcription factors that allow it to give that kidney specific expression
Vs
A liver cell expresses a different population of transcription factors. They’ll allow it to give the liver specific gene expression. …..
Overall, we mix and match these transcription factors in different cells to regulate our genes differently in the different cell types
What is combinatorial control?
It is the mixing and matching of transcription factors to give a cell specific expression
What is the activation domain used for in transcription factors?
It is used to interact with other molecules to help recruit them to the gene for transcription
Ex.
Co-activators
HATs
Chromatin Remodeling Complexes
Mediator
Pre-initiation complexes (complex contains RNA polymerase)
Does the activation domain bind DNA?
No, it binds other things like co-activators Croton, remodeling complexes and hats, etc.
What is needed for the initiation of transcription in eukaryotes ?
We need to make our chromatin look like the 10 nm fiber beads on a string structure
—-so we must open the chromatin structure
——— we do this using a combination of HATs and chromatin remodeling complexes (use energy to push nucleosomes out of the way, freeing up a piece of DNA —- it can remove histone proteins, essentially nucleosomes, which opens up the chromatin structure)
Pioneering transcription factors
What is an example of a chromatin remodeling complex?
SWI/SNF : pushes the nucleosomes out of the way and opens the DNA structure
What is the first thing that we have to do during initiation to open up the chromatin?
A special transcription factor called pioneering transcription factor is going to come in and it’s going to bind to DNA in this very condensed region (we are considering heterochromatin that needs to be opened for transcription)
The activation domain of these pioneering transcription factors will then help recruit other transcription factors such as HATs through protein-protein interactions
The HATs will start acetylating the histone tails of the nucleosomes opening up the chromatin structure
The acceleration of the his stone tales will recruit the chromatin remodeling complexes, such as
SwI/ SNF that will push the nucleosome out-of-the-way opening up the DNA specifically a promoter site or an activator site
However, this can happen the other way
Some pioneer transcription factors will recruit chromatin remodeling complexes after they bind to DNA
These chromatin remodeling complexes will then remodel the chromatin, and then that will help recruit our HATs
So, we need both HATs and chromatin remodeling complexes (SWI/SNF) to do this
What happens next after we create the 10 nm fiber beads on a string structure with hats and chromatin remodeling complexes?
Your activators will bind to a site like the enhancer site
Then activators through their activation domain, they’re going to help recruit other proteins to this site :
Co-activators
Other transcription factors
Chromatin remodeling complexes
Mediators (very large complexes)
- a bridge that attaches to all activators and on the other side of it attaches to the RNA Polymerase Complex at the promoter
Once bridge is formed transcription machinery knows that we are good for transcription
How do we recruit RNA polymerase to the promoter especially when it is not able to locate promoters on its own like the prokaryotic RNA polymerase?
General transcription factors are instrumental in bringing RNA polymerase to the promoter
What happens after an activator binds to a promoter proximal or promoter distal region of the eukaryotic DNA?
It will then recruit co-activators including mediators, HATs, and chromatin remodeling complexes through its activation site
What are mediators?
They are large co-activator complexes that interact with activators on one end and RNA polymerase on the other
Just as other co-activators they are not able to interact with DNA directly because they do not have a DNA binding domain
There are about 30 subunits in each of our human mediators
- they are going to interact with activators at promoter distal, and proximal sites binding to parts of them through their amino acids and then it will also buy to the RNA polymerase complex. Add the promoter, therefore bridging the signals between the activators and the race through this direct physical interaction.
This then lets the RNA polymerase know that it is ready to begin transcription because it has lots of positive signaling from all of the activators that are being transmitted through the mediator
Where are the activator binding sites or promoter proximal and distal elements where activators can bind ?
Promoter proximal: TATA box, INr
Promoter Distal: Enhancers
What are mediators?
It acts as a bridge between the activators and RNA polymerase that’s going to be assembled on the promoter
The co-activators that are recruited by the activation domain of the activators will interact with the mediators
This tells the polymerase that we are ready to start transcription
Once chromatin are opened to the 10 nm beads on a string structure what will happen next?
Activators will come in and buy to DNA either at the promoter proximal regions or promoter distal regions
Then those activators through their activation domain will recruit other things like co-activators, HATs, chromatin, remodeling complexes, and a mediator
What is a mediator?
A very, very large co-activator complex
- just like other co-activators they do not bind to DNA (co-activators do not have dna binding domains)
- they bind to activators to help mediate their signals with something else particularly RNA polymerase
-
How many RNA polymerase eukaryotes have and what is RNA Polymerase II?
We have three: RNA Polymerase I synthesizes rRNA
RNA Polymerase III synthesizes tRNA
RNA polymerase II synthesizes mRNA and some non-coding RNA
What do eukaryotic RNA polymerases mimic?
Prokaryotic core RNA polymerase that has two alpha subunits, a beta, a beta prime and omega sub unit
In addition, they have caught four common subunits
Just like the prokaryotic core RNA polymerase, eukaryotic RNA polymerase II does not have a sigma sub unit, which is a part of the prokaryotic holoenzyme therefore it is not able to locate promoters on its own
What is specifically on RNA polymerase II that is not on the other RNA polymerases?
The C terminal domain or the C terminal tail (very necessary for transcription initiation and RNA processing)
- it is disordered (does not have a specific structure)
Why do we call the special transcription factors needed to help the RNA polymerase find the promoter general transcription factors?
Because they are transcription factors required at every RNA polymerase II promoter that is used so wherever RNA polymerase two is transcribing a gene these specific transcription factors are needed
to guide RNA polymerase II to the promoter
So, look for TFII (indicates it is a general transcription factor for RNA Polymerase II) in addition to a letter to specify which one it is
What is different at eukaryotic promoters than prokaryotic promoters?
They do not have a consensus sequence that all eukaryotic promoters are measured against
Every eukaryotic promoter usually has an INR sequence, which is an initiator at +1 (start site)
- one of the sequences that will be recognized in the promoter
Only 10% of our promoters have TATA boxes
There will be a lot of diversity in the promoters Purcell, which can have regular regulatory sequences in an INR in one cell, but have a Tata box and INR in another cell or just have a Tato box and regulatory sequences and yet another cell
How does the complex of general transcription factors recognize promoters regardless of so much diversity and their promoter sequences?
The very first general transcription factor TFIID is the part of the complex that is going to read the DNA and find those promoter sequences
—- it is made up of 14 different sub units, all of them have DNA binding domains that recognize different sequences
——-one sub unit is called Tata binding protein (TBP)
——-13 other sub units are called TBP associated factors or TAFs
-TF2D is going to mix and match. It’s different sub units to find whatever promoter sequences are at our promoter.
once TF IID locate the promoter sequences, then we will have additional proteins or general transcription factors of this complex come in
— so next will be TF2A, then TF2B and then finally TF2F will bring RNA polymerase to join the complex that is forming at the promoter by binding to TF2B
After RNA polymerase two is brought to the promoter by TF2F what happens next?
TF2H (aka dna helicase) comes in, the full pre-initiation complex which is also the closed complex
So now we have to have activators binding to promoter proximal and distal sites, along with co-activators, recruited HATs , and recruited chromatin remodeling complexes as well as the mediator to bridge all of their signaling to this RNA polymerase complex by connecting with RNA Pol 2 via the C Terminal Domain (CTD) of RNA Pol II and the TFIIH
RNA Pol II will wait for the ‘green flag’ which is the phosphorylation of its C terminal domain (CTD) by the kinase portion of TFIIH which does not just have the function of helicase
- once mediator binds to TF2H it will know that the bridge is formed and we have lots of positive signaling meaning we’re ready for transcription then it will initiate phosphorylation of the CTD of RNA Pol II
TF2H is going to use ATP to separate the strands of DNA by unwinding the DNA and making the transcription bubble
Then RNA Pol two is going to transcribe everything from the transcription start site through the coding sequence continuously until it gets to the terminator site where transcription will end
True or false the elongation step of transcription is identical in prokaryotes and eukaryotes
True
What about negative gene regulation in eukaryotic, gene expression or transcription?
Professors combined to the activator binding sites and compete with them that means the activator cannot bind to DNA nor stimulate transcription
We also have core pressors that act similar to activators … so they will bind to activator molecules and prevent anything else from interacting with those activators therefore, we will not be able to recruit hats, chromatin remodeling complexes, other co-activators or mediators, and therefore we cannot do transcription
Repressor can block the RNA plane race pre-initiation complex from forming or the processor combine and then act as a docking site for our HDAC’s —-they will remove the acetyl group from the lysine of a histone protein, which will then enable that lysine to interact with other histone proteins of neighboring nucleosomes , which will then lead to the condensing of the chromatin structure
—-after this, the chromatin will still be in a euchromatin state
If we want to put the chromatin into a heterochromatin state, then after removing the acetyl groups, we need to methylate histones to get heterochromatin through hyper methylation ( we are therefore taking an already condensed euchromatin and making it more condensed to form heterochromatin)
——-so in order to form heterochromatin, we start with using the enzyme Histomethyltransferase which will methylate the histones. Once the histones are trimethylated, then another enzyme called heterochromatin protein 1 (hp1) will then bind to the trimethylated histones which act as docking sites for it then the HP1s will interact with each other further increasing the number of interactions between the nucleosomes and further condensing the chromatin structure When that happens then they recruit more HMT‘s, which will methylate more histones and then recruit more HP1s that will bind and interact and cause more condensation of the chromatin….. this will spread throughout a certain region, which will form a really condensed heterochromatin structure.
True or false post transcriptional modifications happen in both prokaryotes and eukaryotes
False they only happen in eukaryotes because prokaryotes do not process the RNA that’s produced from transcription there RNA that is produced from transcription is the same mRNA that is used for translation which is different from eukaryotes where we do have these post transcriptional modifications to form the mRNA that we will use for translation
Which level is the overall gene expression or a gene product of a cell regulated at?
It is regulated at many levels, but the level that regulates it the most is at the transcriptional level
What are the two things that we will add onto the primary transcript once it is produced from transcription?
We are going to add a five prime cap on the five prime end of the pre MRNA and we will add a poly A tail to the three prime end of the pre mRNA
Then we will remove the introns (non coding sequences of DNA) and splice together the exons
Also, we will retain the 5’ UTR (untranslated region between the promoter and 1st exon) and the 3’ UTR after the last exon - these are not being translated into protein, but they have regulatory sequences inside them which tells translation when to start and stop
What enzyme adds the five prime cap onto the RNA molecule?
The cap synthesizing complex
-it will bind to those phosphorylated serines on RNA polymerase then it will wait for the five prime end of the RNA molecule to come out of the polymerase
The cap is a 7-methyl guanosine
- guanosine nucleotide with an extra methyl group on the 7 position
The 7-methyl guanosine will be attached to the 5’ end of your RNA
-all RNA molecules will have three phosphates at its five prime end
Is the seven methyl guanosine cap encoded in the gene?
No, it is added on post transcriptionally
Why do RNA molecules have three phosphates at its five prime end?
RNA primary can synthesize RNA de novo meaning without a primer so it will take one nucleotide triphosphate and another nucleotide triphosphate and make a phosphodiester bond between them. Then it’s gonna start adding on nucleotides to the three prime end of the second nucleotide.
—— since that nucleotide at the five prime and was never used to make a phosphodiester bond. We never removed a pyrophosphate so that means that all nucleotides at the five prime end of our molecules still have three phosphates attached to them and therefore all RNA molecules have three phosphates at their five prime end.
What is the first step in putting the methyl cap of the five prime end of RNA polymerase?
Removing the gamma phosphate from the five prime end
After removing the gamma phosphate from the five prime end during placement of the methyl cap, what are the remaining steps that the capping enzyme does?
- Step two is putting a GMP from a GTP onto the five prime end of the RNA and in the process it will release a pyrophosphate (proves G is added post-transcriptionally)
This is all the capping enzyme does…
add’l enzymes add methyl groups…one of them adds a methyl group to make 7 methyl guanosine
What is the purpose of the five prime methyl cap?
- It is going to protect the RNA from nucleus degradation…. therefore it protects RNA from EXOnucleases which cannot degrade it with the cap on.
- The cap is going to interact with other proteins that are involved in exporting the RNA out of the nucleus and proteins involved in translation
overall, it is important to mediate future downstream processes
**remember that as soon as the five prime end of RNA comes out from the RNA polymerase, will grab it adding on the methyl cap **
True or false addition of the poly A tail is directly tied to transcription termination
True
Once RNA polymerous transcribes the termination sequence of the RNA will it stop or keep going
?
It will keep going
But when the termination sequence is transcribed into the RNA, there’s going to be additional proteins that are bound to that c terminal domain (CTD) of RNA polymerase 2
What are the additional proteins that bind to CTD of RNA polymerase two once transcribes the termination sequence of RNA
?
They are factors called polyadenylation factors or termination factors
They recognize the termination sequence in the RNA
so when that sequence comes up in the RNA, those polyadenylation factors are going to bind to it
—-binding to the termination sequence will stimulate the endonuclease activity of these factors
—— endonuclease is going to cut a phosphoric bond in the RNA somewhere downstream from the termination sequence …..
What happens when the endonucleases from the polyadenylation factors release the RNA?
this releases the RNA molecule, but the factors are still bound to that termination sequence in the RNA.
—-
They will stay on the RNA in order to add the poly a tail
What is poly a polymerase ?
A polyadenylation factor
…
poly a polymerase is going to bind to the three prime end of the RNA molecule and it will use it as a nucleophile to add a bunch of A’s onto the three prime end ~ 80 to 250 A’s
This process is directly tied to transcription termination
Why doesn’t poly a polymerase use a template strand?
Because the only thing I can do is add A’s to the RNA
What is the point of the poly a tail?
- It has similar roles as the methyl cap prevention of nuclease degredation
- To help export the RNA out of the nucleus.
- And there will be proteins that interact with it that are needed for
Translation
What happens when we remove the Introns (denoted by A, B, C, D…) and slice together the Exons (1,2,3,4,….)?
We create a continuous coding sequence, a.k.a. a continuous open reading frame
Will the Exon’s always retain their order five prime to three prime despite how the splicing occurs?
Yes, they will
They will never be rearranged in the five prime to three prime direction
How does the cellular machinery determine where the beginning of an Intron is and where the end of an intron is?
They determine where the entrances are based on a rule that we call the GU/AG rule the GU is at the beginning of the intron and the AG is at the end of the intron
What is the phosphodiester bond between the G at the beginning of the Intron and the last nucleotide of the Exon?
5’ splice site
Likewise, at the end of the Intron, the phosphodiester bond found between the AG of Intron and and the nucleotide of the next Exon is called the
3’ splice site
We called these splice sites because we are going to break these bonds through the process
Why is the branch point A important for removing the intron and where is it located?
It is really important because it is going to act as the nucleophile for this reaction
What is going to be catalyzing the process of reading the GU/AG bonds and then breaking the 5’ and 3’ splice sites (phosphodiester bonds)?
The spliceosome (made of many subunits)
What are two components that every subunit of the spliceosome has in it?
Protein (used for structural purposes) and RNA (used for function “business end”)
These are called small nuclear ribonuclear proteins (snRNP) complexes “SNURPs”
How many snurps make up the spliceosome?
Five of them and each one is named after the small RNA molecule that’s inside of it
Ex. U1 is the RNA molecule in U1 snRNP
What do each of the snurps do?
U1 Snurp: binds to the five prime splice site in RNA after finding it
It tells spliceosome where the 5’ splice site is located
U1 base pairs to GU at the beginning of the Intron and the surrounding sequence….so it finds that site by base pairing
Everything outside of structural purposes is being done by the RNA molecules in the snurps
Explain the spliceosome reaction at the macromolecule level…
- Spliceosome will be recruited to the site
A. U1 will bind to 5’ splice site to define it
B. U2 will define branch point A by using the sequence from its RnA molecule to base pair to the sequence surrounding Branch point A
C. U5 comes in to organize everything
D. U4 & U6 come in together as a U4/U6 complex (U6 has the catalytic site for solicing reaction and U4 blocks its ability till its needed) - The spliceosome is inactive until it is rearranged to the active form of the spliceosome by U4 leaving and U1 going into the catalytic site of U6
- Break the phosphodiester bond at the 5’ splice site
- Break the phosphodiester bond at the 3’ splice site
- Remove intron
- Glue (splice) the exons together forming a normal phosphodiester bond between them
explain the spliceosome reaction at the nucleotide level
We have two transesterification rxns (therefore breaking one ester bond and forming another in a different position)
—— we do this twice for the phosphodiester bonds
- We are going to use the 2’ sugar on the branch point A as the nucleophile by attacking the 5’ end of the intron ( the 5’ phosphate of the G at the beginning of the intron)
—— this will form a unique 2’ - 5’ phosphodiester bond between the G and the A - The 3’ hydroxyl group at the end of the exon will be the nucleophile for the second transesterification reaction by attacking the 5’ phosphate at the end of the other exon which will form a 3’ - 5’ phosphodiester bond between them (this is what glues the 2 exons together)
3 (simultaneously with 2) During the process of creating the 3’ - 5’ phosphodiester bond, we break the bonds between the end of the Intron and the beginning of the second Exon
What does alternative spicing allow us to do?
It allows us to make multiple different proteins from the exact same gene
So cells can make different versions of a protein from the same gene to meet their needs
So alternative slicing is slicing in different ways to result in different final mRNA molecules (95% of our genes use it)
Depending on which promoter you use ( because eukaryotic genes have multiple promoters) we would get a different RNA molecule
We can have multiple termination sites in a gene that results in a different RNA and a different protein as well
What tells the spliceosome where to splice (gluing exons together) the mRNA?
RNA binding proteins
They bind to sequences in the RNA and regulate the process of splicing
Splicing activators -SR proteins (serine/arginine rich) - promote splicing by binding to sequences in the exons called exonic splicing enhancers (ESEs) ….. through binding to sites on these they tell the spliceosome to splice there
Splicing repressors - Heterogeneous nuclear ribonuclearproteins (hnRNP) bind to sequences in the exons called exonic splicing silencers (ESS) , and then they inhibit splicing at that site….. tells spliceosome not to use splice site at that site
What do the cells do with SR proteins and HNRNP‘s?
They expressed different populations of both sets of proteins by mixing and matching them to give the splicing patterns that that specific cell type needs
What is the signal that we are ready for translation?
Once we have the cap from the cap binding complex on the five prime end and the poly a tail from the poly a binding protein on the three prime end and the SR proteins for enhancement of various exons and HNRNP‘s for repression of other Exon’s …… once all these proteins are coding the RNA ……
Then we are ready for translation
Next, we have to get the RNA out of the nucleus and into the cytoplasm where our translation machinery is located
How do we conduct nuclear exporting of mRNA out of the nucleus and into the cytoplasm?
- Cap binding protein will bind to the nuclear pores in our nuclear membrane and it will thread out the RNA in a five prime to three prime direction
- Cytoplasmic versions of the proteins that prepared the mRNA for translation will bind to the RNA and they will promote the process of translation.
