transcription Flashcards
list 3 ways that RNA polymerases are different from DNA polymerases
RNA pol adds ribonucleotides (U instead of T), doesn’t require a primer, lacks 3’-5’ exonuclease proofreading
T or F: RNA pol requires a primer to work
false; it doesn’t need a primer
T or F: the polymerization method is the same for DNA pol and RNA pol
true
describe the polymerization mechanism for RNA pol
nucleophilic attack by the 3’ OH on the phosphorous of an incoming NTP. Pyrophosphate exits as NMP is incorporated into the growing RNA strand. Mg2+ facilitates the nucleophilic attack + the other helps displace the leaving PPi
in which direction is the template strand copied
3’-5’
describe the general shape of the bacterial RNA pol
5 core subunits in a claw-like shape
what does the sixth subunit of bacterial RNA pol do + what is it’s name
sigma subunit: binds transiently to the core and helps direct it to specific DNA binding sites
T or F: RNA pol only binds to DNA in the presence of the sigma subunit
true
list the components of the holoenzyme
core + sigma subunit + DNA
where on the DNA will the sigma subunit bind during bacterial initiation
it binds to promoters upstream of the genes that need to be transcribed
which two promoter sequences does the sigma subunit bind to in bacterial initiation
-10 and -35
why are the -10 and -30 called consensus sequences (ie why consensus specifically)
bc these sites are usually similar across different promoters
describe why promoters establish a basal level of gene expression for every gene that can be upregulated or downregulated
mutations in promoters can greatly lower RNA pol binding and transcript initiation
what happens to the DNA when sigma binds to the consensus sequences in the promoter in bacterial initiation
DNA bends
what happens once sigma binds to promoter and DNA bends? (ie what is the result of this)
allows the RNA pol to interact with both the -10 and -35 regions at the same time
in bacterial initiation, describe what occurs once RNA pol is bound to both -10 and -35
the upstream promoter (UP) binds to the core RNA pol
the UP is rich in which bases
AT
T or F: in bacterial initiation, the upstream promoter (UP) binds to the sigma subunit
false; it only binds to the core RNA pol, not the sigma subunit
bacterial initiation: describe the events once everything is bound (consensus sequences, RNA pol, UP)
sigma binds to promoter and brings the polymerase core with it. First a closed complex is formed where DNA is still wound, then an open complex is formed when DNA begins to unwind near -10
bacterial initiation: where does DNA begin to unwind
-10
when bacterial initiation has started via conformational changes in RNA pol, what happens once RNA pol clears the promoter
sigma dissociates and NusA protein binds in its place
bacterial elongation: once sigma dissociates, which protein binds in its place
NusA
which step in the process signifies the start of bacterial elongation
once RNA pol has cleared the promoter, we’re now in the elongation stage
why is bacterial regulation tightly regulated
if RNA pol falls off too early, it has to restart the transcript all the way back at the promoter
what two types of bacterial termination are there
rho-dependent and rho-independent
in rho dependent termination, what is the name of the sequence that RNA has
RUT sequence
what does RUT stand for
Rho utilization element
in rho dependent termination, describe how the rho protein interacts with the RUT sequence on RNA
it binds to the RUT and travels 5’-3’ down the RNA
is RUT on RNA or DNA
RNA
which way does rho travel down the RNA after binding to RUT
5’-3’
which bases is RUT rich in?
C and A
what type of activity does rho have
helicase
T or F: rho is ATP-dependent
true
at which point will rho separate DNA/RNA
when it runs into the elongating polymerase
describe how rho-independent termination occurs in bacteria
a region of DNA produces an RNA transcript that has self-complimentary sequences hairpins with itself = strain. This disrupts base pairing in the RNA-DNA hybrid section and RNA pol can no longer bind properly to the RNA so it falls off
in rho independent termination, where is the hairpin located in regards to the desired termination site
10-20 nucleotides before
in rho dependent termination, describe the structure of the hairpin near the 3’ end
has 3 highly conserved U residues
list 3 key differences between pro and eu transcription
- the 3 RNA pol have similar subunits but each is recruit to different types of promoters
- eu promoters have much greater diversity and only two conserved promoter sequences (TATA box)
- eu has no sigma molecule: instead basal TFs bind to the promoter
what is the position of the TATA box
-30
what does CTD stand for
carboxyl terminal domain
which molecule is the CTD located on
RNA pol II
which RNA pol is the CTD located on
RNA pol II
describe the structure/orientation of the CTD on RNA pol II
it projects away from the remainder of the complex and is spatially separated from it by an intrinsically disordered linker
the tail includes a 7 aa sequence that repeats 25+ times
which in the first TF that arrives at the promoter
TFIID
structure of TFIID?
multisubunit complex, one of which is TATA-binding protein (TBP)
what does TBP stand for
TATA-binding protein
on which TF is TBP located
TFIID
the whole TFIID complex reaches across ___ elongated base pairs
70
what does TBP do
binds to the TATA box
result of TBP binding to the TATA box?
this positions the initiator sequence in the center of the elongated DNA with TFIID straddling it on either side
what happens after TBP is bound to the TATA box and the initiatior sequence is in the center of the elongated DNA
TFIIB binds to TBP
what does TFIIB bind to
TBP
what does TFIIB recruit
RNA pol II
where does TFIIB recruit RNA pol II to?
to the promoter
which TF is RNA pol II tightly associated with
TFIIF
once RNA pol II and TFIIF are recruited to the promoter, what happens
TFIIE binds to pol II
what does TFIIE recruit
TFIIH
what is the name for the complex once all TFs and RNA pol II have bound to the promoter
the closed preinitiation complex
which TF has helicase activity to start unwinding DNA
TFIIH
eu: near which site is DNA unwound
near the initiator site
what is the name of the structure that forms after TFIIH unwinds DNA
the transcription bubble
what is the name of the complex of unwound DNA + TFs + RNA pol II
open initiation complex
which event starts transcription
TFIIH kinase activity: phosphorylated CTD of RNA pol II and the promoter is cleared
once the promoter is cleared in eu initiation/elongation, which TFs leave the complex?
TFIIH and TFIIE leave
which TF stays tightly associated with pol II during elongation
TFIIF
binding of which type of molecules prevent pol II arrest in eu elongation?
elongation factors
describe what happens at the end of eu transcription
elongation factors leave and termination factors bind
what happens to RNA pol II after eu transcription is over
it’s dephosphorylated and recycled
what happens if the eu transcription machinery halts at certain DNA lesions
TFIIH can recruit NER machinery (nucleotide excision repair)
TFIIH mutations can cause which disease
xeroderma pigmentosum
T or F: the phosphorylation status of CTD changes throughout transcription
true
treatment of tuberculosis involved what type of actions from an antibiotic
an antibiotic can stall the RNA pol II at the stage of promoter clearance, preventing it from ever entering elongation
what molecule is the 5’ cap
7-methylguanosine
purpose of the 5’ cap?
help protect mRNA from ribonuclease degradation in the cytosol
by which linkage is the 5’ cap attached to mRNA
5’-5’ triphosphate linkage
describe the 5’-5’ triphosphate linkage that links the 5’ cap to the mRNA
condensation of GTP at the exposed 5’ phosphate end of mRNA. two 5’ carbons are thus involved in the linkage with 3 P’s in the middle
when is the 5’ cap added
in early transcription: after the first 20-30 nucleotides
what two complexes are required for the addition of the 5’ cap
cap-synthesizing complex and cap-binding complex
T or F: both the cap-synthesizing and cap-binding complex can interact with the CTD
true
T or F: the protection provided by the 5’ cap is permanent
false; it’s not permanent
describe why the 5’ cap’s protection isn’t permanent
the cytosol has cellular decapping enzymes, so eventually the cap will be removed and the transcript will be vulnerable to 5’-3’ exonucleases
what are the 3 splicing mechanisms
group 1, group 2, spliceosomal
describe group 1 splicing
free guanosine 2’ or 3’ OH nucleophilic attack on the phosphorous between exon and intron. The 3’ OH of the displaced exon then does a nucleophilic attack on the other end of the intron
describe group 2 splicing
the nucleophile for the first attack is an adenosine WITHIN the intron. This forms an intermediate lariat structure
T or F: group 1 and 2 introns are self splicing (no protein or ATP required)
true
T or F: spliceosomal introns require large RNA-protein spliceosome complex
true
T or F: ATP is not required to splice spliceosomal introns
false; ATP is required for spliceosome assembly
describe the structure of the spliceosome
made up of snRNPs
describe the structure of snRNPs
each one has a small nuclear RNA (snRNA) and many proteins
list the five snRNAs that are involved in splicing
u1, u2, u4, u5, u6
T or F: the snRNAs have intrachain binding and complimentary regions to parts of the mRNA transcript
true
which sites on the mRNA mark splicing sites
GU at the 5’ end of an intron and AG at the 3’ end of an intron
which snRNA binds to the 5’ GU of an intron
U1
which snRNA binds to internal A of an intron
U2
how is the inactive spliceosome formed
u1 binds to GU, u2 binds to internal A, then u4-u6 dimer and u5 join to make the inactive spliceosome
what occurs once the inactive spliceosome is formed
spliceosome is rearrnaged to displace u1 and u4. It also places u6 at the 5’ intron end to make the active spliceosome
describe the structure of the active spliceosome
u1 and u4 have been displaced, u6 is at the 5’ end of the intron and the internal A and GU are super close together now
what events occur once the active spliceosome has been formed
u2 which is bound to the internal A and u6 brings the A close enough for nucleophilic attack on the GU (via u6)
which snRNA is the catalytic one
u6
what happens once u6 does nucleophilic attack on GU
lariat formation + intron release
which two snRNAs are tethered to the CTD on RNA pol at the end of splicing
u2 and u1
where does the intron go after being spliced
stays in the nucleus
what happens to the intron in the nucleus after being spliced
it’s degraded
which conserved sequence is found at the end of a transcript
AAUAAA
describe the addition of the poly A tail
RNA pol II synthesizes RNA beyond the AAUAAA sequence. Endonucleases cleave RNA downstream of AAUAAA, then adenylate polymerase adds 80-250 As
in poly A tail addition, where do the endonuclease + polyadenylate polymerase come from?
AAUAAA is bound by a protein complex bound to RNA pol II CTD that includes these two enzymes
benefit of alternative splicing?
more than one protein can be produced from the same primary transcript
describe polyA site choice
poly A tails can form at different sites
list some ways you can get alternative splicing
- alternative exons, 5’ sites or 3’ sites
- mutually exclusive alternative exons, alternative promoters and first exons, alternative poly A site and terminal exon
