Cell And Molec Final Flashcards
What are the levels of chromatin packing? (5)
- nucleosomes -> 30nm chromatin fiber -> looped domains -> heterochromatin -> highly condensed duplicated chromosome of a diving cell
What is a nucleosome? (What is it made up of)
- histone octamer protien is wrapped with DNA (twice) and then “sealed” or held together with the H1 histone (looks like a little pill)
What is a solenoid?
Nucleosomes start to coil around each other (create a spiral) in one long chain
how are looped domains formed?
The solenoid strand binds to the protein scaffold at differing intervals, creating the loops
What determines what genes are expressed?
Where the loops of solenoid attaches to the protein scaffold
How is heterochromatin formed?
The protein scaffold loops around itself (creates a coiled coil) along with the looped domains
Give a general overview of how DNA is packaged and chromosomes are formed.
DNA is wrapped around histone proteins and held together with a H1 histone protein to create a nucleosome. Then those nucleosomes are strung together, creating a solenoid. The solenoid is a long strand which is then bound at different intervals along the protein scaffold. Then the protein scaffold coils around itself creating a coiled coil, which is called heterochromatin. Once at this point, it can continue to condense itself, creating a highly condensed duplicated chromosome of a dividing cell (this is the shape we recognize as a chromosome)
What state must chromatin be in for a chromosome to form?
A condensed state
Why are chromatin condensed into a chromosome?
They are easier to transport
What does a nuclear pore do?
Detects whether a protein has the correct sequence and allows it into the cell through the nuclear membrane
What are the factors that determine the length of the cell cycle? (Give an example of each)
- age (new cell will replicate faster than an older cell)
- species (some species cells divide quicker like bamboo)
- tissue type (nerve cells take really long whereas gut cells don’t take long at all)
- temp **ONLY IN LAB (higher temp is slightly faster replication)
What is the order of the cell cycle?
GAP 1 - SYNTHESIS PHASE - GAP 2 - MITOSIS
What is interphase?
The time of G1, S, and G2 before mitosis (the preparation of the cell to divide)
What happens to the cell during G1 phase (gap phase I)? What is this doing (what’s the purpose)
- cell grows, organelles are all duplicated, and building blocks are added
- the purpose is to prepare for S phase and eventually division
What happens during Synthesis phase? (S phase)
The nucleus (and the DNA) and the centrosome is duplicated
- this is where transcription/translation take place
What happens during the second gap phase? (G2)
-cell grows more in size
- prepares the cell for mitosis
What are the “building blocks” that must be made in G1 phase in order for DNA replication to occur
- nucleotides
- DNTPs
-enzymes
-ribosomes
What is the role of kinase in the cell cycle?
Kinase catalyzes phosphoryl transfers from ATP to substrates
Go through each step of the cell cycle (including each of mitosis) and show whether they are 2n, 4n, or just n. What is the result?
G1 (2n) - S (4n) - G2 (4n) - P (4n) - M (4n) - A (4n) - T + C (2n)
- results in 2 2n cells
What are the phases of mitosis?
Prophase - Prometaphase - metaphase - anaphase - telophase
What happens during prophase in mitosis?
Chromosomes move to either side of cell and spindle fibers form,
What happens during Metaphase in mitosis?
Nuclear membrane breaks apart, the spindle fibers attach to the chromosomes and they line up at the equator (m- middle) (chromosomes are still together in X)
What happens during anaphase?
The spindle fibers shorten, and the centromere divides so that each chromosome is split into their 2 separate chromatids
What happens during telophase?
Nuclear membranes form around each set of chromosomes, they spread back out and the spindle fibers break down
What happens during cytokinesis?
The cell membrane pinches between the 2 nuclear membranes creating a cleavage furrow, and eventually separates the 2 cells into 2 identical daughter cells
What is meiosis?
The cell division of gametes
What are gametes?
Sex cells (egg and sperm)
What is the result of meiosis?
4 unique, haploid, daughter cells (n)
How many divisions are there in meiosis?
2
Why are there 2 divisions in meiosis but only 1 division in mitosis?
Gametes are haploid cells, you need to split it twice (1 4n -> 2 2n -> 4 n). Regular body cells are diploid, meaning you only need 1 division ( 1 4n -> 2 2n)
In what phase does transcription and translation take place?
Synthesis Phase (S phase)
What happens during prophase 1 in meiosis I ?
Synapsis: when homologous chromosomes line up and wrap around each other super tight creating the possibility for homologous recombination (crossing over)
What are homologous chromosomes?
The same chromosome of each parent ( moms chromosome 1 and dads chromosome 1 line up together, they’re homologous)
What is crossing over
When pieces of DNA is swapped between homologous chromosomes
- rearranges homologous chromosomes
What does crossing over ensure?
Variability
When does independent assortment occur? (What phase)
- metaphase I
What is a chiasma?
The structure that physically links the homologous chromosomes during meiosis
What is independent orientation?
The order in which the chromosomes line up during metaphase I
- for example you have AB and CD, they can line up 4 ways : AB CD, BA CD, AB DC, BA DC
What is the formula for independent orientation?
2 ^n where n is the number of chromosome pairs
What happens during metaphase I in meiosis?
- spindle fibers move to either pole and the homologous chromosomes line up in the middle
- ## when independent orientation takes place
What happens during anaphase I during meiosis?
The homologous chromosomes are pulled apart, leaving 4 separate chromosomes
What happens in telophase I during meiosis?
A nuclear membrane is formed around each set of chromosomes and the nuclei are split
What happens during cytokinesis I during meiosis
the cell membrane divides into 2 separate cells
What is the result of meiosis I?
2 unique diploid cells ( 2 2n)
What happens during prophase II in meiosis?
- no more crossing over, they begin to line up
What happens during metaphase II during meiosis II?
The chromosomes all line up in the middle of the cell and the spindle fibers attach to the centromeres
What happens during anaphase II during meiosis II?
The centromeres are pulled apart, each half of the chromosomes pulled to the other side
What happens during telophase and cytokinesis II?
The nuclear membrane forms and separates the chromosomes and then the cell membrane closes in in the middle and splits the cells apart
What is the result of meiosis II?
4 unique haploid cells
List all the differences between meiosis and mitosis
- meiosis is meant to be variable while mitosis is supposed to be exact replication
- meiosis results in 4 gametes (unique) and mitosis results in 2 identical (clone) daughter cells
- meiosis results in haploid gametes, mitosis results in diploid somatic cells
- cross over and synapsis are common in meiosis while being very rare for mitosis
- chromatids separate in anaphase in mitosis but they don’t separate until anaphase II in meiosis
- meiosis has 2 divisions while mitosis has 1
Draw the structure of thymine
Draw it!
Draw the structure of Cytosine
Draw it
Draw the structure of adenine
Draw it
Draw the structure of guanine
Draw it
Draw the structure of uracil
Draw it
What are 2 purines?
Adenine and Guanine
What are the 3 pyrimidines
- uracil thymine and cytosine
What is the difference in structure between uracil and thymine? How does this allow for them to be interchangeable?
The only difference is the H3C attached to the 6th carbon (thymine) vs just H (uracil)
- the N that the base uses to bind to the surfer of the backbone is undisturbed and therefore can still bind whether it is a cytosine or a uracil
Why are purines more sensitive to pH change?
When pH decreases specifically, depurination occurs
What is depurination
N-glycosidic bonds are cleaved to release the corresponding adenine or guanine from DNA
What does the term “anti parallel” in dna structure refer to?
The 2 DNA strands run opposite each other, one runs 5’ -> 3’ and one runs 3’ -> 5’
Where do bases attach? (Which end)
- 3’ end
Which bases have 3 bonds between each other and which bases have 2 bonds?
- there are 2 bonds between A and T or A and U
- there are 3 bonds between C and G
Draw how A and T and C and G bind together (draw the H bonds)
Draw it
Which bonds will break easier / with less energy (AT OR CG)
- AT bc there are less bonds to break
Are hydrogen bonds destroyed when exposed to heat
No
If DNA is denatured by heat, and then cooled slowly, what will happen to the base pairs? What if they were cooked quickly?
They will come back together in the correct pairing/order
If cooled quickly; they may not come together in the correct order
How is DNA replicated? ( type of replication )
Semi-conservative replication
What is the difference between conservative and semi conservative replication
- conservative replication is where the DNA replicates and the 2 old halves are in the same cell and the 2 new halves are in the same cell
- semi conservative is when the DNA replicates, half of the new and half of the old DNA go in one cell and the other halves go into the other cell
What is the biggest difference between bacterial (prokaryotes) replication and eukaryote replication?
The origins of replication - eukaryotes have multiple at the same time, where bacteria only have 1
Can eukaryotes replication bubbles be bidirectional? Can prokaryotes?
Yes, both can be bidirectional
What are the limitations of DNA polymerase?
- it can only travel 5’ -> 3’ (only can attach to 3’ hydroxyl
- it can not synthesize DNA “from something new”
- it must have a primer, but cannot make its own
- it cannot umwind or hold the DNA open for replication itself
If you put : template DNA, DNA polymerase, and dNTPs into a test tube, will new DNA be created?
No
- you need to also add primers and “additional things”
What are topoisomers? What do they do?
- relaxes DNA in order to open it up, and cuts it
Where does DNA polymerase bind?
The origin
Why do eukaryotes need multiple replication sites ( whereas prokaryotes only need 1? )
- eukaryotes DNA is too long, it would take too long to replicate with just 1 site it needs multiple
Which strand is synthesized continuously?
The leading strand
What are Okazaki fragments
Pieces of the lagging strand replication (lagging strands is replicated in fragments
Which direction does the leading strand replicate?
Into the replication fork, from 3’ to 5’
Which direction does the lagging strand replicate
Out of the fork, 5’ to 3’
What role does ligase have in dna replication?
The ligase attaches the phosphate to the hydroxyl
What is the overall direction of replication ?
Into the fork, 3’ to 5’
What is “codon usage” ?
some codons are used more than others, depending on species/kingdom
What is the start codon?
AUG : methimine
How many stop codons are there? What are they?
3 :
- UAA
- UAG
- UGA
How many base pairs make up a codon? How many codons make up an amino acid?
- 3 base pairs create 1 codon
- 1 codon translates to 1 amino acid
List some causes of substitution mutation
- radiation
- chemicals
- UV radiation
- X Rays
- viruses
- gamma rays
what is the difference between a normal red blood cell and a sickle cell ?
- a normal hemoglobin DNA strand would code for “GLU”, which causes a hydrophilic polypeptide
- in a sickle cell, the DNA has a substitution, making it code for “VAL”, causing a hydrophobic polypeptide. This Alters the quaternary structure
How does substitution affect the gene?
- if the base pair is substituted and the corresponding codon is not the same as the original, this will lead to a differen amino acid made
- if the base pair is substituted but the corresponding codon is the same as the original was supposed to be, no errors are made
How does base deletion affect the gene?
Base deletion would cause a shift in the reading frame, most likely coding for the wrong codon
How does insertion affect the gene ?
Insertion causes a shift in the reading frame, most likely causing a change in the codon
What is a nonsense mutation?
When a codon thay typically codes for an amino acid, codes for a stop codon instead
- UAC becomes UAG
What are missense mutations?
Alter one amino acid for another
- changed amino acid, doesn’t crest stop codon
What are frameshift mutations?
- insertion or deletion, when the reading frame is shifted
What are null mutations?
When there is a change in the DNA, codon, mRNA, and AA but chemically similar AA is replaced
What are silent mutations
Mutation but it doesn’t cause a change in the amino acid
- CUU instead of CUC - but they both code for Lec
The trombone model is proposed in what types of organisms
- prokaryotes
What is the trombone model
- idea of how replication takes place in bacteria
- helps coordinate DNA synthesis between the two strands
- suggests the presence of a third polymerase to help DNA synthesis
- proposes that the lagging strand forms a loop, so that the leading and lagging strand replication proteins can contact one another
What does Helicase do?
Breaks the hydrogen bonds between the nucleotide based and unwinds the DNA at the replication fork
How does DNA begin replication, if the DNA polymerase cannot start on its own?
- RNA primase
What does DNA ligase do?
Joins together the Okazaki fragments on the lagging strand
What does topoisomerase do?
Nicks and unwinds strands to release mechanical stress of unwinding
How do the DNA strands stay open during replication?
Single-strand binding proteins (SSB proteins) bind to a single stranded DNA at the replication fork, keeping it open for replication
What does DNA polymerase I do?
- removes RNA and replaces with DNA
- editing enzyme
What does RNA primase do?
- initiates new strand synthesis
What does RNA polymerase do
It is an enzyme that makes short pieces of RNA
What makes primers?
Primase (enzyme)
- a type of RNA polymerase
What attaches Okazaki fragments together
DNA ligase
How are RNA primers removed (replaced with DNA)
- DNA polymerase I
What are the similarity(s) between RNA polymerase and DNA polymerase
- they both polymerize 5’ -> 3’
What are the differences between DNA polymerase and RNA polymerase
- DNA : used for replication
RNA : used for transcription - DNA : cannot seperate or hold strands open without help (helicase, topoisomerase, SSB)
RNA : can separate and hold strands itself, does not need additional enzymes - DNA: no de novo ( cannot start transcription on its own)
RNA : de novo synthesis (can start transcription on its own) - DNA : very fast (800 nt/s)
RNA : slow ( 20-40 nt/s)
What is the role of promoters?
They are DNA sequences before genes that provide a starting point for RNA polymerase in order to initiate translation
- mark the start of the gene and help bring it in for activation
How does RNA polymerase work
- using a DNA template, the rna polymerase builds a new rna molecule through base pairing
- the RNA polymerase binds to the promoters (specific initiation sites on the DNA), and unwinds the DNA just enough to start de novo synthesis
What is a promoter?
A DNA sequence that tells the RNA polymerase where to start
Why is there space between the promoter and when translation starts (about 25-35 bases)
RNA has to recognize the sequence in order to start translation, must sit between the sequences in order to recognize
RNA polymerase can only add nucleotides to which end?
The 3’ hydroxyl end
Where is the TATA box located
Upstream from the transcription unit
What determines the length of the promoter sequence
The gene that is being translated
How many base pairs between the TATA box and +1
Between the -10 and the +1 is about 10 or so base pairs
What do each of the RNA polymerases in Eukaryotes recognize?
- RNA polymerase II : recognizes genes, has TATA box at about the -25
- RNA polymerase I : recognizes tRNAs
- RNA polymerase III : recognizes most ribosomal rnas
How does termination in prokaryotes and eukaryotes differ?
- prokaryotes : either rho dependent or rho independent mechanisms
- eukaryotes: a poly A single and a downstream terminator sequence
What is the difference between Rho independent and Rho dependent termination? What type of cells experience this type of termination?
- prokaryotes
- rho dependent requires rho protien to bind to the rut site to stop
- rho independent creates a hairpin loop like structure to terminate transcription
What is the hairpin loop like structure
- happens in rho independent
- a consecutive series of bases rich in C and G that pulls together to form a loop - create a weak point where the rna breaks off
List the differences between prokaryotic mRNA and eukaryotic mRNA. What is similar about them?
- similar : 5’ and 3’ untranslated regions
- PRO : usually polycistronic
mature after being transcribed
can be both transcribed and translated at the same time - EU : not usually polycistronic (monocistronic)
Not mature after being transcribed
cannot be transcribed and translated at same time
Why can’t eukaryotes transcribe and translate at the same time if prokaryotes can?
In eukaryotes, transcription happens in the nucleus and translation happens in the cytoplasm whereas in prokaryotes, it all happens in cytoplasm
What does polycistronic mean?
- the mRNA has 2 or more cistrons
- 2 + proteins are coded on a single molecule of mRNA
What happens during the maturation of eukaryotic mRNA?
- attachment of a 7 methylguanosine cap to the 5’ end
- introns excision (spliced)
- formation of 3’ end by cleavage and addition of non-templates poly(A) tail
What does the small nuclear RNA do?
Acts as a binding site for the 2 ends of the intron
What is splicing
2 ends of the intron bind to the small
Nuclear RNA, where it is spliced and the exons are ligated together
Without the Lariat structure, what could happen
Intron could potentially be translated, creating a toxic sequence
What 3 things do you need for translation to happen?
- an mRNA transcript
- a ribosome (rRNA)
- charged tRNA (amino acid attached = charged)
Why should the tRNA and the mRNA have corresponding base pairs
To ensure the correct amino acid is made
What is at the 3’ end of a tRNA
CCA sequence with an ester bond to an amino acid
Are all tRNAs identical?
No but similar in structure
What is the acceptor arm on a tRNA
CCA - 3’
How many arms does a tRNA have
5
What is the function of aminoacyl tRNA synthetase?
- pairs tRNA with the corresponding amino acids in order to decode the mRNA
What is an “activated amino acid”
An aminoacyl tRNA
- tRNA bound to amino acid
What is the brief concept of wobble
- there’s an alternate base pairing between the anticodon (of the tRNA) and the codon (of the mRNA)
- wobble always results in the same amino acid
How does wobble benefit tRNA
- allows to reduce the number of tRNA
There’s fewer amount of different tRNA needed to read the same amount of codons (there are 61 but with wobble you need much less)
What is Inosine (I) ?
Post transcription modified purine
UUI anticodon (on tRNA) can code for the codon AAA, AAU, or AAC. This is because of what?
Wobble
Draw the structure of inosine
Draw it
What are ribosomes composed of
Protein and ribosomal RNA
Where does activity occur in a ribosome
Between the protein and the ribosomal RNA
What is the difference in structure between prokaryote rRNA and eukaryote rRNA
- pro : 16S (s)subunit + 50S (l)subunit=70S
- Eu : 40S subunit + 60S subunit = 80S
What type of cells is the Shine-Dalgarno sequence found
- only prokaryotes
What does the shine-dalgarno sequence allow for
Allows the mRNA to bind to the small subunit of rRNA (16S)
How does the rRNA know which initiation code to start at?
It’s the closest/first one (AUG sequence)
What is different about the first amino acid in bacterial translation
It is an altered amino acid
- formyl group attached
What do IF2 and IF3 do in translation initiation?
They act as chaperone proteins to make sure the proteins get to the right destination
- IF2 : chaperones tRNA
- IF3 : chaperones the 30S subunit to help bind to the S-D site
What is IF1 thought to do?
Hold the large subunit and the small subunit apart from each other until they are ready to create the complex
In eukaryotes, what does the small subunit bind to?
- 5’ cap
Without the shine-dalgarno sequence, how do eukaryotes find AUG?
- scanning
- moves down the length of the mRNA until it finds a recognition site
What is the Kozak sequence
- acts as initiation site, it’ll be near the AUG
What is the difference in function between the Shine-Dalgarno sequence and the kozak sequence?
- shine dalgarno : (only pro) is for binding the mRNA and rRNA
- kozak : (only Eu) is a recognition site to find the start codon (AUG)
What happens at the A site during elongation?
- “amino acid site”
- new tRNA (brings new amino acid)
What happens at the P site during elongation
- “protein or polypeptide site”
- newly made polypeptide sits until the new amino acid comes to attach to it
What happens at the E site in elongation
- exit site
- where the tRNA goes after the amino acid was taken off
What does EF-Tu do?
Chaperone protein, brings in second tRNA
What does EF-G do?
- motor protein that moved the mRNA down a triplet to be read
What is translocation in elongation
The movement of the mRNA to be able to read other triplet (codons)
How does the termination of translation occur? (Eukaryotes) ?
The stop codon codes for a release factor, causes the whole strand to fall off along with all other subunits
What does the signal-recognition particle (SRP) do ?
- targets signal peptide-breaking proteins to the prokaryotic plasma membrane or the eukaryotic ER membrane for secretion or membrane insertion
What does the SRP protein complex bind to in translation, what does it do
- binds to ER signal sequence, blocks translation
If a polypeptide has an internal stop transfer sequence, where will the protein be released?
- into the cytosol
If a polypeptide has an internal start transfer sequence, where will the protein be released?
- into the ER lumen
T/F : all genes are regulated at some or more levels
True
What are constitutive genes?
- genes that are continually expressed
- “housekeeping genes”
What are regulated genes
- genes that control cell growth and cell division
- expression is regulated by the needs of the cell and the environment as needed
- NOT continuous
What are the 2 categories of genes?
- regulated genes
- constitutive or “housekeeping” genes
What is the most common type of regulation for prokaryotes?
Transcriptional regulation
What does transcriptional regulation control?
Whether the mRNA is made or not
How does transcriptional regulation work?
- it controls the access of the RNA polymerase to the promoter
What are the 2 types of regulations
- negative
- positive
Describe negative regulation
- an inhibitor or repressor (protein) is involved (whether the repressor is turning on or off)
How is positive regulation controlled?
- an activator
What is an activator in regulation
- a protein that aids in the process of transcription - turns the genes on
Can negative regulation turn genes on and off? Can positive?
- negative can turn genes on or off but positive almost always turns them on
What does procesivity mean?
- it means the enzyme can catalyze consecutive reactions without releasing the substrate
RNA polymerase is (processive/recessive)
DNA polymerase is (processive/recessive)
- they are both processive
What is an operon?
A cluster of genes in which expression is regulated by operator-repressor protein interactions, operator region, and the promoter
What are the contents of an operon?
- promoter
- repressor
- operator (controlling site)
- coding sequences
- terminator
What is an inducer
A chemical or environmental agent that initiates transcription of an operon
What is induction
The synthesis of gene product(s) in response to an inducer
Do transcription and translation occur before or after induction
After only
What was the first operon discovered
- Lac operon
What does the lac operon break down
Beta-galactosidase
- glucose can be used directly but galactose needs to be broken down further
If there’s no lactose present, what will happen during prokaryotic regulation
The repressor binds to the operator and blocks that site from binding
If lactose is present, what will happen in prokaryotic regulation?
The lactose will bind to the repressor, changing shape (conformation) and making it fall off the operator site allowing for regulation to occur
If there is a mutation on the operator site (Oc mutation), what will happen? (Prokaryotic regulation)
- the repressor cannot bind to the operator site, therefore regardless of the presence of lactose, the gene will be on all the time- the gene will be constitutive
If there is a mutation in the repressor, what will happen? (In pro regulation)
- the repressor cannot bind to operator site, creating a constitutive gene regardless of the presence of lactose
If there is an I-S mutation in the repressor what will happen? (Pro regulation)
- the mutation alters the repressor so that lactose cannot bind, therefore the inducer doesnt work
- causes the repressor to bind to the operator and not come off
Is catabolite negative or positive regulation?
Positive
If there is glucose but no lactose present in a bacterial cell, what will happen to the cAMP concentration? Will there be Lac transcription?
- low cAMP
- NO lac transcription
If there no glucose and no lactose present in a bacterial cell, what will happen to the cAMP concentration? Will there be Lac transcription?
- high cAMP
- NO lac transcription
If there is glucose and lactose present in a bacterial cell, what will happen to the cAMP concentration? Will there be Lac transcription?
- low cAMP
- no lac transcription
If there is no glucose but there is lactose present in a bacterial cell, what will happen to the cAMP concentration? Will there be Lac transcription?
- high cAMP
- YES LAC TRANSCRIPTION
What is the only condition that lac transcription will occur? Why is this? (Bacterial cell)
- will only occur when there is no glucose present, only lactose
- this is because bacteria prefer glucose as an energy source
What does the catabolite activation protein (CAP) do?
- helps (stabilizes) the RNA polymerase bind and stay on and allow it to go through transcription
What typically determines the ability of CAP to bind
- the availability of (cofactor) cyclic amp (cAMP)
What is the only condition (glucose and lactose concentrations) that lac transcription will take place
- when there’s no glucose, only lactose
- cAMP will be high
- turns lac operon on
Is catabolic activation (the use of CAP) positive or negative regulation? Why?
- CAP
- it stabilizes the RNA polymerase for transcription to occur, helps transcription, no repressor present
When tryptophan concentration is low, is the is the trp operon turned on or off?
- On
- low trp creates more trp, therefore turning it on
Is the trp operon positive or negative regulation?
- negative bc there is a repressor being used
In the trp operon, what is considered an active repressor?
When an aporepressor and a co-repressor bind
Eukaryotic gene expression is controlled much more tightly than prokaryotic; what are specifics that eukaryotes look for?
- time
-location - dosage
Why is eukaryotic gene regulation much more complicated than prokaryotic?
- because eukaryotes are usually multicellular and so much more complicated in development
What are the 4 levels that eukaryotic regulation takes place?
- pre-transcriptional (whether transcription will happen or not)
- transcriptional (during transcription)
- post-transcriptional (translational) (what happens to the mRNA once transcribed)
- post-translational (the protein)
Do prokaryotes have an operator? Do eukaryotes?
- only prokaryotes have an operator
What are CPG islands?
A series of C and G that comes right in front of a gene
What do the CpG islands do?
They can be (heavily) methylated in order to suppress a gene
If a CPG is found around the promoter of housekeeping genes (or frequently expressed genes) is it methylated or not methylated?
- not methylated
- when methylated it suppresses the gene
What is a housekeeping gene?
- genes essential for general cell functions
When DNA is methylated, how does its structure change?
It goes from an open conformation to a closed confirmation
What is being acetylated during DNA methylation?
Histones
If methyl groups are added (methylation), and acetyl groups are removed (deacetylation), what happens?
- if acetyl groups are removed, the structure will collapse (because they help keep it open)
- the methylation will help it collapse further
If a region of DNA is destined for silencing, what happens?
Histone deacetylases, and ATP-dependent chromatin remodelers being the gene silencing process
What’s a big factor in whether genes are turned on or off
Environmental factors
What is the basis of epigenetics?
DNA methylation and histone acetylation
What does the open or closed conformation depend on?
- the topology of the DNA / whether it is available and transcribable
What is the core promoter
Where RNA polymerase binds
What are upstream response elements (URE)?
A Sequence upstream an where you’ll have a transcription factor (protein)
If there is an activator protein involved, is it considered positive or negative regulation?
Positive regulation
What does a response element do?
Binds either a repressor or activator
If the response element binds an activator, what will happen
The activator will stabilize the core promoter and allow transcription to take place
If the response element binds a repression transcription factor?
- the repressor will destabilize the core promoter, transcription is inhibited
What is the difference between repressor son prokaryotes and repressor son eukaryotes
- In prokaryotes the repressor blocks the operon from binding (gets in the way)
- eukaryotes the repressor completely destabilizes the protein
How do repressors bind to a helix-turn-helix motif
Like a paper clip
How does the repressor bind in a zinc-finger motif
The repressors are held with a “finger like structure”
- the zinc ion is held in place by different amino acids
What would happen to the zinc finger motif if there was a mutation that took away the negative charge of amino acids
The whole thing would fall apart because that is what the zinc binds to creating the finger like structure
What are the 4 structural motifs that are proposed for the DNA binding domains of eukaryotic transcriptional regulator proteins?
- helix-turn-Helix
- zinc finger
- leucine zipper
- helix-loop
The leucine zipper is the location where what happens ?
Proteins are held to each other
What does the helix-loop-helix motif consist of?
- 2 alpha helices separated by a loop of amino acids
What is transactivation?
The increased rate of gene regulation
If no transactivation factor binds, will transcription happen? What if it does bind?
- transcription will happen either way, but will happen faster with transactivation factor is bound
What is transinhibition
- when an inhibitor binds, it destabilizes the polymerase
There are only about 25k genes but so many more proteins, how is this?
Alternative mRNA splicing
Describe alternative mRNA splicing
If you have an mRNA with the exons : A B C D
you can have a protein that is ABC or ABD or ABCD, all creating different proteins from 1 section of mRNA
What are ARE (AU rich elements)
- the are found at the 3’ UTR and control whether eukaryotic mRNA will be degraded
-more ARE, the quicker the degradation occurs
What controls the stability of mRNA in eukaryotes
- AREs or AU rich elements
A longer poly A tail, the more or less stable?
The longer the polyA tail the more stable
What are miRNA
MicroRNA
- short mRNA sequenced used to regulate mRNA
What do dicer proteins do?
Further cut the mRNA into a single strand (no more loop)
If the cell is in trouble, what will occur in translation? (Eukaryotic)
Translation will stop, “SOS function”
- phosphorylation of elF2 (eukaryotic inhibition factor) and translation inhibition
When elF2a is phosphorylated, how does that stop translation in eukaryotes?
The auto phosphorylation of elF2a changes the conformation and allows it to fund to the active elF2B and makes it inactive
What are Homodimers
2 of the same subunits
What are heterodimers
2 different proteins bound to each other
What are the 3 ways that transcription can be regulated in eukaryotes pre-transcription
- DNA methylation
- deacetylation
- chromatin structure
What are the 4 ways that transcription can be regulated in eukaryotes during transcription
- transcriptional factors
- response/control elements
- transactivation
- response elements
What are the 4 ways that transcription can be regulated in eukaryotes post-transcriptional (translational)
- alternative splicing
- mRNA stability
- miRNA inhibition
- alteration of translational factors
What are the 3 ways that transcription can be regulated in eukaryotes post-translational
(regulating the protein)
- dependence on a secondary effector
- binding of protein subunits
- chemical post-translational modification
What are the 3 methods of examining gene expression
- Northern (RNA) Blot
- western (Protein) Blot
- RT-PCR (reverse transcriptase PCR)
What does northern blot determine
- whether the RNA transcript has been made or not
- therefore determines activity
Give a brief overview of RNA blot
- a specific piece of mRNA is located, make a radioactive phosphorus and make it part of the corresponding dna, then let it sit. They will bind to each other (hybridization), you wash away the excess and use an autoradiogram and X-ray paper
What does the western blot detect?
Whether the protein itself is there or not
What is PCR?
Polymerase chain reaction
What is the purpose of PCR?
AMPLIFY DNA
Enzymatic in vitro replication of DNA
What are the 3 stages of PCR? How many times does the DNA go through these stages?
- denature (95°C)
- reanneal (50-60°C)
- extension (~72°C)
Repeat ~35x
Give an overview of the PCR process
- melt DNA (90°C)
- anneal primers flanking the region of interest (55°C)
- synthesize complementary strand (72°C)
- repeat
If the DNA strand is too long, by how much will they increase with every repetition of PCR
- they will double
If the DNA strand is just the right size, by how much will they increase with every repetition of PCR
- they will increase exponentially
How does PCR work if the enzymes will be denatured at temperatures like 90°C?
- use the enzyme TAC which is an enzyme that thrives in extreme heat
What could misprinting lead to in PCR?
- the wrong sequence being replicated
What could cause the wrong section of DNA to be replicated during PCR?
Mispriming
How does RT (reverse transcriptase) work?
- it is a DNA polymerase that uses mRNA to make cDNA (complimentary DNA)
What does RT-PCR allow for?
- determine whether the mRNA is being expressed, or to amplify the complementary DNA
If PCR Leads to amplification very quickly, what does this mean? What if it is a slower process?
- a faster amplification means there is a high amount of mRNA present
- a slower increase would be because of a low mRNA concentration
What is DNA microarrays used for
To look at hundreds or thousands of genes at once
