Lec 17 - RNA Synthesis & Processing Flashcards
RNA synthesis
e coli RNA polymerase
how many subunits? synthesizes what? differences from dna?
.. non template/coding strand?
- 5 core subunits
- synthesizes all RNAs: mRNA, rRNA, tRNA, other non-coding RNAs
- core RNA pol can synthesize RNA but cant recognize promoters
- unlike DNA, proofreading 3’ –> 5’ exonuclease
- error rate 10^-4 to 10^-5
- active site split between B and B prime
- only 1 strand of DNA duplex transcribed to RNA; template strand used by RNA pol (transcript seq matches nontemplate strand)
- RNA and nontemplate strand complementary to template strand
usually non-template/coding on top (5’ >3’) & template below (3’>5’)
e coli
sigma factor & promoters
which sigma factor used? promoter binding effects?
sigma factor
- bacterial RNA pol requires sigma factor protein to bind RNA pol + promoter (can transcribe alone, but not initiate)
- sigma factor: σ70 (“housekeeping gene”)
promoters
- diff sigma factors bind to diff promoters
- first base of RNA is +1, bases before/upstream are negative (no 0)
- sequence unimportant; distance important (closer to binding site = tighter bond)
- promoters *more similar to consensus *bind sigma factor with higher affinity (1) recruit more RNA pol (2) lead to more RNA produced
- strong promoters also have UP element that binds core polymerase alpha (notable in rRNA)
major groove & DNA binding proteins
Major groove
- protein binds intact double helix; interactions with major groove of DNA
- proteins can instert alpha helix without side chains disrupting canonical base-pairing
- sequence-specific protein-DNA interactions
DNA-binding proteins
- AA side chains form base-specific favorable interactions that match with h bonding
- diff for each protein
e coli
transcription initiation
gen steps? diff from dna rep how?
- multistep process; double helix is closed…
- core RNA pol + sigma factor bind (form closed complex) & unwind (occurs in bubble within RNA pol)
- template strand unpaired & available for RNA synthesis
- RNA pol begins RNA synthesis without primer* (diff from DNA rep*).. just joins 2 NTPs
- In open complex, 8-15 nt of RNA polymerized
- short RNA released and new copy made (abortive initiation)
- eventually sigma factor released & RNA pol leaves promoter to synthesize full-length RNA (promoter clearance)
e coli
transcription elongation
diff from DNA how?
- after promoter clearance, RNA Pol elongation is highly processive (~100,000 nt)
- no mechanism to restart RNA synthesis after polymerase disengagement/falls off (diff from DNA)
e coli
transcription termination
rho dependent vs rho independent
**Instrinsic (rho-independent) termination: **RNA (& DNA) seq alone stop transcription, no other proteins
- in e coli, RNA stem loop (hairpin) followed by poly-U stretch ends transcription
rho-dependent termination: ATP-dependent helicase rho required
- rut site (req on RNA) with transcription pause sequence downstream
- rho helicase loads at rut site in nascent RNA
- pause allows rho to catch up with polymerase
unknown whether helicase directly interacting with RNA pol, or simply pulling RNA out of the polymerase
transcriptional activators, repressors, regulators
how is transcription affected? mutant effect?
activators
- binds near promoter & increases transcription (inc RNA prod); e.g. by favorable interactions with sigma protein/core polymerase
- loss of an activator/binding site
- mutants that lose activator protein/binding site –> uninducible: not transcribed/ always “off”
repressors
- binds near promoter & decreases transcription (dec RNA prod); e.g. by* physically blocking *sigma factor or core polymerase from binding
- loss of repressor/binding site
- mutants that lose repressor/binding site –> consitutive: always transcribed/ always “on”
regulators
- fine tuning gene expression; promoters can have combo of activator/repressor binding sites
- complicated programs of gene expression control
lactose metabolism
when are cells expressed? lacY & lacZ? minor product of lacZ?
- cells express enzymes for lactose metabolism only when (1) lactose is present & (2) glucose is absent
- allolactose is a minor product of lacZ directly sensed within the cell as an indicator of lactose (upregulates)
- **lacY **encodes permease that brings lactose into cells across the membrane
- lacZ encodes B-galactosidase which breaks down lactose into monosacharides (& allolactose)
lac operon
general info?
repressors bind operators
- LacI repressor protein constitutively (always) expressed from one promoter
- 3 lac operator sites (inverted symmetric repeat seq; binding sites for LacI near lacZYA promoter)
- O1 site bound w/highest affinity; O2/O3 have slightly weaker)
regulation by lactose + glucose
CRP & cyclic AMP
regulation by lactose
- LacI repressor also binds allolactose; allosteric change in allolactose-bound LacI reduces DNA affinity
- … Lac I repressor shuts off lacZYA promoter when (allo-)lactose absent
- … lacZYA promoter unrepressed when (allo-)lactose present
regulation by glucose
- activator protein CRP binds near lacZYA promoter & interacts with core RNA pol (alpha subunit)
- CRP interaction recruits RNA pol to promoter, leading to more transcription (favorable binding > higher affinity > more RNA)
- CRP binds DNA only when bound to cyclic AMP (aka cAMP)
- bacteria make cAMP in reponse to low glucose
CRP-cAMP binding allosterically increases CRP-DNA affinity; CRP binds DNA only when cAMP present (i.e. glucose is low)
lac regulation trends
glucose, cAMP, lactose
- NO LACTOSE = NO expression
- glucose low, cAMP high, lactose present = high gene expression
- glucose high, cAMP low, lactose present = low gene expression
eukaryotic RNA Polymerases
I - III details? what do they transcribe? # of transcripts?
RNA pol I (>50% RNA synthesis)
- pre-ribosomal RNA
RNA pol II (10,000 - 100,000 transcripts)
- all coding messenger RNAs (mRNAs)
- many non-coding RNAs
- comlex & highly regulated initiation
RNA pol III (100s of transcripts)
- transfer RNAs (tRNAs)
- one small ribosomal RNA (5S rRNA)
- various small, non-coding RNAs
eukaryotic RNA Polymerase II
poll II initiation
RNA pol II details? steps of initiation?
similar to bacterial RNA polymerase
- RNA pol II is large & has 12 protein subunits
- pol II initiation is complex & requires diff general transcription factors (multiprotein complexes)
- transcription factors similar to sigma factor in bacteria
1) pol II recruited to DNA by transcription factors
2) transcription bubble forms
3) CTD phosphorylated during initiation; polymerase escapes promoter
4) transcription elongation aided by elongation factors after TFIIE and TFIIH dissociate
- still cycles of abortive initiation and eventually promoter escapes into elongation; pol II phosphorylated before it departs
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written as TFiis
pol II CTD phosphorylation
how many aa repeats? phosphorylation purpose?
- unfolded region at C-terminus of Pol II has multiple 7 aa repeat copies (27 in yeast; 52 in humans)
- this repeat can be modified with phosphorylation (specifically at serine 2 & 5)
- CTD phosphorylation determines what phase of transcription RNA pol II is in
- during initiation serine 5 is phosphorylated; as poll II progresses, serine 2 becomes more & more phosphorylated; and serine 5 becomes dephosphorylated
transcription factors, enhancers, mediators
transcription factors
- euk regulators (activators/repressors) are called transcription factors; seq-specific DNA binding, at a few promoters
- general transctiption factors (TFIIA, TFIIB) function generally in all transcription with specific TFs (Gal4) that function specifically on a few genes; with Pol II at most promoters
enhancers
- euk promoters regulated by distant enhancers, which are activator binding site (can bind hundreds/thousands of bp away)
mediators (allow enhancers to be far away)
- large protein complex bridging activators to RNA poll II
- functions as general TF and is a coactivator
- euk activator proteins usually bind to* mediator complex,* not pol directly of other general TF
