Lecture 5 - Transcription and RNA Processing Flashcards
Do RNA polymerases require a primer?
NOPE
Does nascent RNA remain hybridized to template DNA?
NOPE
Which is more permanent: RNA or DNA?
DNA
Which is more accurate: replication or transcription?
Replication
How frequent are errors during transcription?
1 per 10,000 nucleotides
Are RNA and DNA polymerases structurally and evolutionarily related?
NOPE
Why do many errors during transcription go unnoticed?
Because many RNA strands are synthesized
What are mRNAs? Function?
Messenger RNAs
Code for proteins
What are rRNAs? 2 Functions?
Ribosomal RNAs
- Form the basic structure of the ribosome
- Catalyze protein synthesis
What are tRNAs? Function?
Transfer RNAs
Central to protein synthesis as adaptors between mRNA and AAs
What are snRNAs? Function?
Small nuclear RNAs
Function in a variety of nuclear processes including the splicing of pre-mRNA
What are snoRNAs? Function?
Small nucleolar RNAs
Used to process and chemically modify rRNAs
What are scaRNAs? Function?
Small cajal RNAs
Used to modify snoRNAs and snRNAs
What are miRNAs? Function?
MicroRNAs
Regulate gene expression typically by blocking translation of selective mRNAs
What are siRNAs? Function?
Small interfering RNAs
Turn off gene expression by directing degradation of selective mRNAs and the establishment of compact chromatin structures
What is the function of other noncoding RNAs? 3 of them
Function in diverse cell processes like:
- Telomere synthesis
- X-chromosome inactivation
- Transport of proteins into the ER
How are different types of RNAs synthesized?
By different types of RNA polymerases
Are both DNA strands used as templates during transcription?
Not necessarily
How do we call the DNA strand that is not used as the template during transcription? 3 names
Nontemplate strand = coding strand = sense strand
In what direction is RNA synthesized during transcription?
5’ => 3’
How is the template strand assigned?
Functional assignment: called this only when it is serving as the template, which depends on the type of RNA polymerase used and what direction it moves in which is dependent on the direction of the gene
How to tell the direction of transcription?
As the RNA polymerase moves the RNA gets longer and longer
What is a transcription bubble?
Small strand separation in which the RNA polymerase reads the DNA template strand and synthesizes RNA
How is the nascent RNA released by the RNA polymerase?
Released from a ribosomal complex on the RNA polymerase
What are the 3 types of eukaryotic RNA polymerases and what types of RNA does each synthesize? Which ones accounts for most of RNA synthesis?
I: 5.8S, 18S, and 28S rRNAs (70% of RNA synthesis)
II: mRNA, snoRNA, and some snRNA
III: tRNA, 5S RNA, some snRNA (for splicing and poly(A)), scRNA (SRP = signal recognition peptide)
Which type of RNA is regulated more finely and precisely?
mRNA
What is another name for mRNA?
Pol II transcript
What are the 2 main differences between bacterial and eukaryotic RNA polymerases?
- Bacterial RNA pol requires only 1 single protein (sigma factor) for initiation, vs eukaryotic RNA pol require many additional proteins (general transcription factors)
- Eukaryotic transcriptional initiation must deal with the packaging of DNA in nucleosomes
Where is the promoter located? What are its 2 responsibilities? What does this mean for the sequences?
Sequence of DNA that is 5’ to the initiation site of the gene: the 5’ flanking region, which may also contain enhancers or other protein binding sites
Telling the RNA pol:
- Where transcription is going to start
- In what direction transcription should go
Therefore, the promoter is non-palindromic because DNA sequences that are non-palindromic are orientation-dependent and it is also position-dependent
What are the 3 regions of the promoter?
- Distal region
- Proximal region
- Core region
How is the promoter numbered?
Transcription starts at +1 so the promoter is negatively numbered
What is the role of the elements of the core promoter?
Binding sites for transcription factors associated with the initiation that create a large complex to create the right environment for RNA pol II to bind
What are the 4 elements of the core promoter? Positions? Do all core promoters contain all 4?
- BRE = B response element: -37 to -32
- TATA: -31 to -26
- Inr = initiator: -2 to +4
- DPE = downstream promoter element: +28 to +32
NOPE
What binds the TATA box?
A protein subunit of TFIID called the TATA binding protein (TBP)
What core promoter element is contained in ALL human neuropeptide promoter sequences?
TATA box
What are the 3 elements of the proximal promoter?
- CCAAT box
- GC boxes
- CRE = cAMP response element
What is the element of the distal promoter?
NRE = negative response element
What are the 5 general transcriptional factors needed for initiation in eukaryotes?
- TFIID
- TFIIB
- TFIIF
- TFIIE
- TFIIH
What are the 2 subunits of the general transcriptional factors needed for initiation TFIID and each of their function?
- 1 TBP subunit: recognizes TATA box
- 11 TAF subunits (TBP associated factors): recognizes other DNA sequences near the transcription start point and regulate DNA-binding by TBP
2 roles of general transcriptional factors needed for initiation TFIIB?
- Recognizes BRE
2. Positions RNA Pol at the start site of transcription
2 roles of general transcriptional factors needed for initiation TFIIF?
- Stabilizes RNA pol interaction with TBP and TFIIB
2. Helps attracts TFIIE and TFIIH
Role of general transcriptional factors needed for initiation TFIIE?
Attracts and regulates TFIIH
2 roles of general transcriptional factors needed for initiation TFIIH?
- Unwinds DNA at the transcription start point using ATP to create the transcription bubble
- Phosphorylates Ser5 of RNA Pol C-terminal domain (CTD) using ATP, which releases RNA Pol from other factors at the promoter
Describe the 5 steps of transcription initiation.
- TBP of TFIID binds the TATA box
- TFIIB binds BRE
- TFIIE and TFIIH + TFIIF bound to RNA Pol II with other factors all bind to the DNA
- UTP, ATP, CTP, and GTP are all used for helicase activity of TFIIH and CTD phosphorylation
- Disassembly of most transcriptional factors
Which 2 TAFs recognize and bind Inr?
TAF 1 and 2
What is the role of the phosphorylation of the CTD by TFIIH?
- Release of RNA Pol from promoter region
2. Binding sites for further transcription factors: capping factors, splicing proteins, and 3’ end processing proteins
What are 4 additional requirements of eukaryotic transcription initiation?
- Enhancer binding site for activator protein to regulate the expression rate of a gene
- Mediator (which is bound by the activator protein)
- Chromatin-remodeling complex
- Histone-modifying complex
What is the role of the mediator in transcription initiation? Does it bind to DNA?
Coordinates all of the transcription factors throughout the promoter by binding to them with protein binding domains
NOPE
What is the role of the chromatin remodeling complex in transcription initiation?
Remodels the chromatin to expose the DNA and make it available for a transcription bubble to be created
How does transcription elongation affect the rest of the DNA?
It produces superhelical tension in DNA: the transcription bubble unwinds 10 DNA bps (1 helical turn) so the DNA helix forms supercoils if it has a fixed end or rotate 1 turn if it has a free end
In vivo it has fixed ends so it creates negative supercoiling behind the transcription bubble and positive supercoiling ahead of it
How does negative supercoiling affect helix opening?
It facilitates it
How does positive supercoiling affect helix opening?
It hinders it
When does RNA processing occur?
Co-translational process
What happens during splicing? Overall and 3 steps
Primary RNA transcript is processed to eliminate the introns that have been transcribed:
- Recognition of conserved sequences at intro/exon borders including conserved interior sequences
- Removal of intervening sequences with a lariat and 3’ end outside the loop
- Ligation of mRNA back together
What are the 3 different RNA processing activities?
- 5’ capping
- Splicing
- 3’ polyadenylation
Does RNA processing happen in prokaryotes? Why?
The 5’ capping and the 3’ polyadenylation happen but that’s it because they do not have DNA introns to excise and the mRNA does not have regulatory binding sites
What is the purpose of the 5’ cap? 3
- Aids mRNA nuclear export
- Protects from 5‘ - 3‘ exonuclease degradation in cytoplasm
- Targets transcripts to ribosome for translation
What is the purpose of the 3’ polyadenylation? 4
- Participates in termination of transcription
- Aids mRNA nuclear export
- Protects from 3‘ - 5‘ exonuclease degradation in cytoplasm
- Targets transcripts to ribosome for translation
Are all regions of eukaryotic mRNA coding for a protein?
NOPE
Are all regions of prokaryotic mRNA coding for a protein?
NOPE
What is the main difference between prokaryotic and eukaryotic mRNA?
Prokaryotic mRNA products code for multiple proteins, eukaryotic mRNA products only code for 1 protein
What is the half life of mRNA in bacteria?
~2 min
What is the half life of mRNA in eukaryotes? What does it depend on?
min to 10 hrs: VERY variable
Depends on the presence of the 5’ cap and the length of the poly A tail
Can the length of the poly A tail be regulated?
YESSSS
Describe the process of adding the 5’ cap on eukaryotic mRNA.
- PPPNPNP-DNA = PPNPNP-5’DNA + Pi
- PPNPNP-5’DNA + GTP = GPPPNPNP-5’DNA + PPi (5’ => 5’ triphosphate bridge formation)
- GPPPNPNP-5’DNA + methyl group = CH3-(G+)-PPPNPNP-5’DNA
- CH3-(G+)-PPPNPNP-5’DNA + methyl group = CH3-(G+)-PPPN(-CH3)PNP-5’DNA (methyl is added to sugar on SOME caps)
What is the full name of the 5’ cap?
7-methylguanosine triphosphate
What is meant when we refer to RNA Pol II as an “RNA factory”?
During elongation it brings along all of the necessary components to generate a full length mRNA
How was splicing discovered?
Took mature mRNA and back hybridized it to the DNA and saw that many regions of the DNA were not hybridizing with the mRNA and the looping parts were the introns
What is alternative splicing?
Regulated process during gene expression that results in a single gene coding for multiple proteins. In this process, particular exons of a gene may be included within or excluded from the final, processed messenger RNA (mRNA) produced from that gene.
What is an example of alternative splicing?
Calcitonin gene can either produce calcitonin in the thyroid or CGRP (calcitonin gene related peptide) in the brain (involved in taste formation)
What is RNA splicing conducted by?
The spliceosome, an snRNP (small nuclear ribonucleoprotein containing snRNA)
What is the exon definition hypothesis?
Exons tend to be shorter than introns and thus simpler to define which is how splicing occurs and helps as splice junctions are not well conserved and therefore not that easy to identify
How does the exon definition hypothesis describe splicing?
- SR proteins bind to each exon sequence in the pre-mRNA and thereby help to guide the snRNPs to the proper intron/exon boundaries.
- The intron sequences in the pre-mRNA, which can be extremely long, are packaged into hnRNP (heterogeneous nuclear ribonucleoprotein) complexes that compact them into more manageable structures and perhaps mask cryptic splice sites. It has been proposed that hnRNP proteins preferentially associate with intron sequences and that this preference also helps the spliceosome distinguish introns from exons. However, as shown, at least some hnRNP proteins may bind to exon sequences but their role, if any, in exon definition has yet to be established.
What are SR proteins?
Proteins rich in Ser and Arg
What happens during termination? 4 steps
- Consensus nucleotide sequence (AAUAAA) is recognized by CPSF (cleavage and polyadenylation stimulation factors) bound to 5’ side of cleavage site and CstF (cleavage stimulation factor) bound the rest of the 3’ sequence (GU or U rich)
- Cleavage at CA-OH and polyadenylation
- GU or U rich sequence is degraded in the nucleus
- Poly A polymerases adds the poly A tail
What is the role of poly-A-binding proteins?
They stabilize the poly A tail
What happens to the mature mRNA once it reaches the cytoplasm? What is the purpose of this?
It starts collecting all these proteins (including initiation factors for protein synthesis) that will recognize the 5’ cap and 3’ tail and will interact with each other to form a cyclic structure where the 5’ cap is bound to 3’ tail to ensure that the mRNA is intact
What happens when you decap the mRNA?
Rapid 5’ to 3’ degradation
What happens when you the remove the mRNA poly A tail?
Rapid 3’ to 5’ degradation
What is an example of a cytoplasmic deadenylases? How does it work?
e.g. PARN binds the 5’ cap and starts chewing up the 3’ poly A tail leading to mRNA degradation
What is more likely to happen to mRNAs with longer poly-A tails?
More likely to be translated
How are all genes read?
3’ => 5’
Where and when does binding of SR proteins to exons happen?
This demarcation occurs co-transcriptionally, beginning at the CBC (cap-binding complex) at the 5′ end.
Describe the structures formed by introns packaged into hnRNPs.
Each hnRNP complex forms a particle approximately twice the diameter of a nucleosome, and the core is composed of a set of at least eight different proteins.
What transcription factors bind Inr?
TAF 1 and 2 (subunits of TFIID)
Is the 5’ flanking region transcribed into RNA?
NOPE
