Exam 4 Flashcards
Protein Coding DNA is transcribed into?
mRNA,
Transcription begins the gene expression pathway
Non-protein coding has many different categories of sequences including?
RNA molecules are produced by transcription
Which major kinds of RNA are produced due to Transcription?
mRNA, tRNA, rRNA
what are other types of RNA produced
miRNA, TelomeraseRNA , snRNA
What is noncoding DNA?
DNA that never will transcribe
Function of rRNAs
Protein synthesis
Function of tRNAs
Protein Synthesis
Function of snRNAs
mRNA splicing
Function of miRNAs
Regulation of gene expression
Telomerase RNA
template for the addition of telomeres
Describe the differences between Transcription and replication.
Transcription (DNA-RNA)
* Only one DNA strand is used as a template
* No Primer
* ‘specific’ sequence recognition
* No proofreading
* Use rNTP
Replication (DNA-DNA)
* Both DNA strands are used as
template
* Primer (RNA)
* No ‘specific’ sequence recognition
* * Proofreading
* dNTP
Transcription occurs in which direction?
5’-3’
In RNA synthesis the DNA language is copied into the language of?
RNA
In RNA synthesis transcription is selective so it doesn’t?
copy the entire genome
In RNA synthesis what do regulatory sequences mark?
the beginning and the end of DNA segments to be transcribed
In RNA synthesis gene regulation means?
- Which genes are expressed (transcribed and
ultimately translated into proteins) - How much of a gene is expressed (transcribed) HOW MANY COPIES
- Ultimately this should connect to the protein levels produced
What is the template strand for RNA made of and what is its direction?
Template DNA strand in the 3’-5’ direction
Describe significant aspects of RNA polymerase and how it adds building blocks for the formation of a new RNA strand.
-RNA polymerase adds bases on the 3’OH just like DNA polymerase
-nucleophilic attack by the 3’OH on the alpha phosphate with the PPI being released
-Magnesium plays an important role in the chemical binding of rNTPs
RNA polymerase has a great conservation between?
3 kingdoms of the organism
-especially conserved in the active sites
-multiple subunits
Bacteria have how many RNA pol and DNA pol?
Bacteria have only a single RNA polymerase (in
contrast they have 5 DNA polymerases)
Eukaryotic cells have how many RNA pol and DNA pol?
Eukaryotic cells have three RNA polymerases (I, II, III) (in contrast 15 DNA polymerases)
What is the most well-studied Eukaryotic Pol?
Pol 2 ( shape like a crab claw)
How does RNA polymerase employ a two-metal ion catalytic mechanism?
Only one Mg2+ is tightly bound in the active site; the second one is brought in with each nucleotide
RNA polymerase is made up of how many subunits?(Bacteria)
4 subunits
Which polymerase is involved in the transcription of all protein genes( mRNA)?
RNA polymerase ll
What do the other two classes of polymerase transcribe? RNA pol 1 and RNA pol 3
only RNA genes
* RNA polymerase I is located in the nucleolus,
transcribing rRNA genes except 5S rRNA.
* RNA polymerase III is located outside the nucleolus, transcribing 5S rRNA, tRNA, U6 snRNA and some small RNA genes.
Does RNA polymerase need a primer?
RNA polymerase does not need a primer but does recognize specific sites
Does RNA remain on the template strand?
RNA does not remain base-paired to the template DNA; multiple RNA polymerization events at the same time
How accurate is Transcription?
Transcription is much less accurate, mistakes are similar to DNA polymerase without proofreading
Is Transcirpiton broad-like replication?
Transcription is a selective process while DNA replication must occur completely and only once per cell division.
What is the use of a start codon?
for translation, not transcription
Why is the lack of proofreading in RNA polymerase, not a problem?
- RNA polymerases lack a separate proofreading 3′→5′ exonuclease active site, which exists in many DNA polymerases.
- A mistake in the RNA molecule is less consequential than one in DNA
- All RNAs are eventually degraded and replaced
Why do viruses have high rates of evolution?
-in part by the RNA-dependent RNA polymerases (RdRp) that replicate their genomes. Unlike many DNA polymerases, RdRp does not have proofreading activity and is thus unable to correct mistakes during replication.
-These high evolutionary rates have been attributed to the large population sizes, short generation times, and high mutation rates of viruses. Mutation rate, specifically, is an important determinant of evolutionary rate.
what does mutation rate mean in the context of a virus?
mutation rate is the rate at which errors are made during replication of the viral genome.
Retroviruses
like all RNA viruses, exhibit a high mutation rate. Polymerization errors during DNA synthesis by reverse transcriptase, which lacks a proofreading activity, is a major mechanism for generating genetic variation within retroviral populations
Promoters
DNA sequences that exist at the start of the gene
aspects of Transcription
- Recognizing Promoters
- Unwinding DNA
- Transcribing one DNA strand
- Terminate transcription
- RNA polymerases do the job: use no primer, use rNTPs and they need help from other proteins called txp factors or sigma factors: RNA polymerization is processive
- Initiation, elongation, termination
How is the template strand accessed in transcription?
The DNA duplex is unwound for about 17 bp, forming a bubble, which enables RNA polymerase to access the template strand. DNA supercoiling occurs both in front of and behind the transcription bubble.
How does RNA polymerase achieve elongation and termination by?
RNA polymerase binding at the promoter—requiring sigma (σ) factor and, in eukaryotes, transcription factors (TFs)— leads to initiation of transcription by the polymerase holoenzyme, followed by elongation and termination. Once elongation begins, RNA polymerase becomes a highly efficient enzyme
Describe the 5 steps of Transcription at Pol 2 promotors
- Pol 2 recruited to DNA by transcription factors which leads to the binding of RNA polymerase core to the DNA promoter (pre-initiation complex)
- Formation of the transcription bubble (initiation complex)
- Phosphorylation of CTD during initiation
- there is a promoter clearance where there is then elongation (elongation complex)
- Transcription terminates and CTD is dephosphorylated(Termination complex)
rho Independent termination
mRNA sequences form a hairpin, which is followed by three U’s, stalling the polymerase and separating it to form the mRNA
rho dependent termination
there is a rho helicase that separates the polymerase
Inhibitors of transcription
- Actinomycin D (Streptomyces soil bacteria): block transcription elongation in both prokaryotes and eukaryotes
- Rifampicin: prevents promoter clearance in bacteria only; does not affect eukaryotes used to treat tuberculosis and leprosy
Where do antibiotics come from
Some species rely on transcription inhibitors for natural biodefense. For example, the mushroom Amanita phalloides produces α-amanitin
alpha-Amanitin inhibits which of the RNA polymerase?
Pol 2
What is one obvious difference between bacteria and eukaryote transcription?
IS BACTERIA HAVE A SINGLE RNA POLYMERASE ENZYME FOR SYNTHESIZING ALL THE RNA MOLECULES IN THE CELL, INSTEAD OF THE THREE RNA POLYMERASES FOUND IN EUKARYOTES
Initiation of transcription has interplay between RNA polymerase and?
activator and repressor proteins
most common sigma factor?
sigma 70
Describe how sigma factors play a role in bacteria.
-binding to RNA polymerase and DNA sequences
function :bringing the RNA polymerase to the promoter
-helps RNA polymerase in separating DNA stands
sigma 70
- Housekeeping (default) initiation factor
- Helps Polymerase position at +1 to start
- Binds sequences at -35 and -10
What region does the Sigma 70 factor recognize?
-10 and -35 sequences
Abortive initiation
in which shot RNA transcripts are synthesized and released,
until the promoter site is cleared.
How do we study promoters?
-Link promoter regions of interest to reporter genes
o Structural gene and regulatory regions can be separated
o Attach regulatory region to reporter gene: 3 types of reporter genes:Beta Gal , Luciferase, GFP
reporter protein
Is one that you can detect easily and is not present normally in your research system
Give some examples of reporters.
-B-galactosidase , encode by lacz( blue precipitate with Xgal)
-Luciferase from lighting bugs( light from ATP)
-GFP from jellyfish
Lac Z assay
By studying the regulatory sequence you can determine when Lac Z is transcribed it will eventually lead to the formation of b-galactosidase deeper the blue the more b-gal is expressed due to the promoter.
Luciferase assay
Promoter region allows luciferase to transcribe and lead to light expression
GFP assay
regulatory sequence allows for the GFP to transcribe and lead to a fluorescent protein that can be detected.
Transcriptional control in general most often regulation occurs at which step?
Initiation
DNA sequences found in transcriptional control regions interact with?
transcription factors
binding of transcriptional activators or repressors can be?
over 2000 different TF( eukaryotes)
general or specific
What polymerase binds to the promoter region?
RNA polymerase
true or false there is self-transcriptional regulation of transcription factors
true
What happens to protein when there is an activation or repression of mRNA?
activaiton- protein forms
repression- protein is not formed
What does the chromatin look like when the gene is “off”?
condensed chromatin
What does the chromatin look like when the gene is “on”?
Deconsded chromatin which leads to the introduction of mediators, general transcription factors, activators, and RNA polymerase to be present
What are the polymerases of Eukaryotes for transcription?
RNA polymerase I, II, III
TRUE OR FALSE . Transcription factors are NOT efficient at transcription at promoter sequences?
False
In Eukaryotic Transcription what other control exist?
extrinsic and intrinsic all mediated through transcription factors
Give some examples of extrinsic factors
endocrine, metabolic. and osmotic systems
give some examples of intrinsic factors
binding of transcription factors and cofactors
What have recent studies of differentiated cells shown about the commitment of a certain cell type?
the ability to convert them to another cell type simply by manipulating the expression of the transcription factors
embryonic stem cells have the potential to become various cells name those types of cells
Mesoderm
-b and t cells
-macrophages
endoderm
-exocrine cell
-endocrine cell
ectoderm
-astroglia
-inhibitory neuron
-excitatory neuron
What is Transcription Factors?
-DNA binding protiens
-Repressive or activate
General transcription factors
necessary for the assembly of transcriptional machinery
specific transcription factors
specific to certain cells or certain signal or developmental stages
TATA box binds to TBP
is necessary for positioning of the RNA polymerase
Where do promoter elements and enhancers exist?
There are downstream promoter elements as well as upstream elements and also far-away enhancers
How is nomenclature for transcription factors indicative of the RNA polymerase?
TF factors are numbered I, II, III to correspond to the polymerase I, II, III
Eukaryotic Transcription: Multiple Transcription control elements are?
- TATA, BRE, DPE Initiator are part of the proximal promoter; in addition, within 500 base pairs other transcription factors can bind
- Enhancers
Enhancers in Eukaryotic Transcription
➢ Enhancers can be kilobases away
➢ Sometimes works even when inverted
Enhancers and Proximal promoters are similar how?
- Both types are often cell-type-specific
- Some elements can be located in downstream exons/introns or even downstream from final exons
Important Regulatory Sequences (core promoter for Polymerase II)
- TATA box (Binds TBP, TATA binding proteins)
- BRE (TFIIB recognition element)
- DPE (Downstream promoter element)
- TFII (transcription factor II Correlates with Polymerase II)
TATA box function
- 25-35 base pairs upstream
- A TATA box (TATAAA) is necessary for positioning the RNA polymerase II for transcription
- All RNA polymerases use TBP (TATA binding proteins) which need TAFs (TBP- associated factors)
What happens if changes occur at the TATA box
- A single nucleotide change can make a big difference in transcription efficiency (in the crucial region)
- If the region between the TATA box and the start site is deleted, the start site shifts
Introns role in trancripiton?
they allow for splicing which can determine which portion of RNA will become fully mature and expressed to later before proteins. Keep in mind that introns will never be transcribed or reach a state of maturity and only exist as pre-RNA
What role does the mediator play in pol 2 during transcription?
Mediator helps to bridge distant proteins bound to enhancer sequences and Pol II and its general transcription factors, bound near the transcription start site.
Why are Distant enhances important and what role could they play in transcription?
- Far away (up to 50 kbp), common in eukaryotes and very rare in bacterial genomes
- First discovered enhancer, SV40
- 366 base pairs long
-It can actually be inserted in either orientation and can stimulate transcription from all mammalian promoters that have been tested - Can be thousands of base pairs away
Specific transcription factors can exist like CREB and SP1 what was so special about SP1?
- SP1 is a specific transcription factor
- It activates SV40 viral transcription
- It does not activate adenovirus transcription
- Experiments Done in HeLa Cells
- Purified SP1 transcription factor
- Monitor the amount of RNA produced at different concentrations
- Addition of Spl stimulated early transcription of the SV40 DNA 40-fold, whereas transcription of adenovirus DNA was inhibited 40%. This finding suggested that Spl is involved in promoter selection and is not merely a stimulatory general transcription factor
Eukaryotic cells have 3 types of RNA polymerases.
Pol I and Pol III transcribe genes encoding rRNAs and small functional RNAs such as tRNA, respectively. Pol II transcribes protein-coding genes to make mRNA
UTR
The untranslated region of RNA sits between the transcription start site and the start codon( methionine)
The transcription site and translation site don’t always match up with each other ATG is usually downstream.
When looking at a DNA sequence what region does not get transcribed?
The negative portion never gets transcribed only things +1 downstream the start site.
How does termination of Transcription work in eukaryotes?
- Poorly understood but may involve an exonuclease that cleaves mRNA downstream from polyA tail addition
- Pol II termination is coupled to 3’end processing of precursor mRNA transcripts (recognition of the AAUAAA site)
Transcription is coupled to?
DNA repair; RNA processing; mRNA transport are more efficient for genes that are actively transcribed
Describe Transcription coupling with DNA repair.
DNA repair (TFIIH for example can also act in the process of excision repair) repairing DNA template 3’-5’
-* Mutations of TFIIH can cause diseases associated with xeroderma pigmentosa (photosensitivity and tumor susceptibility)
Describe Transcription coupling with RNA processing.
RNA processing (some proteins needed for transcription are also needed for 5’ end RNA processing)
-Removing introns
-adding poly A tail
-5’ end cap
Describe Transcription coupling with mRNA transport.
mRNA Transport out of the nucleus
Name some of the small molecules that regulate the expression of bacterial genes via DNA-binding repressors/activators
Lac operon
* cAMP
* Lactose/allolactose
Trp Operon
* Tryptophan
Lac Operon: What happens when no lactose is around?
doesn’t waste energy making enzymes that break it down
Lac Operon: What happens when both lactose/allolactose and glucose are around?
prefers glucose(different metabolic pathway)
Lac Operon: True or False lactose has to be present in larger amounts than glucose.
True this allows for the mediation of lac transcription
Lac repressor
bind to lac operator region around +11
CAP
Catabolite activator protein binds to the CAP binding site
sigma70 in lac is in which region
-35 and -10
Which lac allows entry of lactose via the protein galactoside permease?
lac Y
B galactosidase converts lactose to galactose and glucose while also making small amounts of lactose to allolactose by which lac?
Lac z
which molecule is the lac operon inducer?
allolactose
Describe the small molecules’ effectors role in the Lac operon
*like allolactose,IPTG( isopropyl beta d-1 thigalactopyranoside) can
bind the Lac repressor and cause its dissociation from the operator, inducing transcription of the lac operon.
*However, IPTG is not a substrate for β-galactosidase. The β- galactoside X-gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside) does not induce expression of the lac operon, but it does serve as an experimentally useful substrate for β-galactosidase, producing a blue color when metabolized.
Inducer or substrate: Allolactose , IPTG , X-gal
Allolactose: both
IPTG: Inducer only
X-gal: substrate only (produces blue precipitate)
Lac Repressor binds to?
operator
what does CAP bind to?
CAP site ( needs cAMP the more of it the better for transcription)
sigma 70 and RNA polymerase binds to?
Promoter
Explain the extra level of complication that occurs with the Lac repressor
- Lac repressor (tetramer) actually binds two sites at the same time (each dimer binds to an operator).
- Secondary binding sites increase the local concentration of lac repressor in the vicinity of the primary operator; this is translated into more available repression
- Remove O2/O3; reduce repression by 70- fold
glucose high, cAMP low, lactose absent?
No gene expression
glucose low , cAMP high, lactose absent?
No gene expression
glucose high, cAMP low, Lactose present?
low level of gene expression
glucose low, cAMP high , lactose present?
High level of gene expression
what mediation role does cAMP have in Lac operon?
levels of cAMP can dictate if there is going to be high or low gene expression, the Lac operon is dependent on both glucose to lactose presence but also the amount of cAMP available.
What’s the relationship between glucose and cAMP?
they have an inverse relationship when glucose is low the cAMP expression will be high
What are the 3 key successes of the Lac Operon
-Sigma70 binding is not perfect( weak promoter vs strong promoter)
-cAMP is generated when glucose falls below a certain level(sensor for glucose concentration
-CAP/CRP-cAMP together greatly stimulate transcription levels
control mechanism in biotechnology
Some vectors have control mechanism for transcription engineered into the plasmid which will better facilitate cloning or control of expression. One of the best-known control mechanisms is the lac operon (an operon is a group of genes). ONLY turned on when you need it.
Once you have cloned a gene you can express protein describe some of the uses of this
- For therapeutic use
- For research use
- Examples
- Insulin
- Growth hormones
- Ribonucleases, transcriptases, polymerases
-Use E.coli cells as factories
Trp Operon
- If tryptophan is low, activate genes that synthesize it
- Trp repressor is transcribed and translated from a gene upstream of the operon that it controls
- When tryptophan is around, trp repressor binds tryptophan and represses transcription of the genes necessary for the synthesis of tryptophan
- Regulatory loop based on the presence or absence of the product (tryptophan)
trpL contains the
attenuator
How does Attenuation work?
by coupling transcription to translation
How does attenuation function work in prokaryotes?
Prokaryotic mRNA does not require processing and since prokaryotes have no nucleus translation of mRNA can start before transcription is complete. Consequently regulation of gene expression via attenuation is unique to prokaryotes
Attenuation occurs based on ?
tryptophan concentration
explain how levels of TRP impact transcription levels
At low levels of trp full lenght mRNA is made, at high levels transcription of the trp operon is prematurely halted
Describe the difference between attenuation and repression in TRP
trpR repressor decreases gene expression by altering the initiation of transcription, while attenuation does so by altering the process of transcription that is already in progress. While the TrpR repressor decreases transcription by a factor of 70 attenuation can further decrease it by a factor of 10
The most stable secondary structure of trp leader mRNA
Attenuation depends on the ability of regions 1 and 2 and regions 3 and 4 of the trp leader sequence to base-pair, forming hairpin secondary structures. The 3-4 hairpin structure acts as a transcription termination signal.
When tryptophan is scare the ribosome stalls allowing a 2-3 hairpin to form.
The ribosome stalls when it encounters the two tryptophan (Trp) codons because of the shortage of tryptophan-carrying tRNA molecules. The stalled ribosome blocks region 1, so a 1-2 hairpin cannot form.
Instead an alternative 2-3 hairpin is created, which prevents formation of the 3-4 termination hairpin. Therefore RNA polymerase can move on to transcribe the entire operon.
When tryptophan is plentiful the ribosome continues, allowing the 3-4 transcription termination signal to form
The moving ribosome complete translation of the leader peptide and pauses at the stop codon, blocking region 2. As a result, the 3-4 structure forms and terminates transcription near the end of the leader sequence.
The Attenuation region gets transcribed to produce ?
non protein coding DNA but serves more as a extra level of control for proper expression of TRP when needed.
