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transcription Flashcards

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1
Q

making RNA from DNA template

A

transcription

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2
Q

how many RNA pol are in bacteria and archaea vs eukaryotic

A

1 in bacteria and archaea

5 in eukaryotic

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3
Q

name the eukaryotic RNA pol and their function/ genes they transcribe

A

RNA pol I- ribosomal RNA (rRNA) (in nucleolus)

RNA pol II- mRNA, small regulatory (siRNA and miRNA)

RNA pol III- tRNA, 5s RNA, snRNA (SNERPs)

RNA pol IV and V- plants only- siRNA

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4
Q

describe the structure of RNA pol in bacteria, archaea, and eukaryotic

A

the basic core structure is HIGHLY CONSERVED b/t bacteria, archaea, and eukaryotic

bacteria- looks like a jaw, has alpha CTD and NTD

archaea and bacteria- look like a hand, eu more related to archaea

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5
Q

describe bacterial RNA pol

A

core enzyme has 5 subunits (~400 kDa)

  • 2 alpha subunits- required for assembly, interacts w/ regulatory proteins.
  • beta subunit- linking nucleotides together
  • beta’ subunit- binds templates
  • omega subunit- helps assembly, chaperone protein
  • jaw like structure with beta and beta’ for the jaws
  • 2 alpha and omega subunit are the at the base, base of cleft is enzyme active site
  • alpha subunit has CTD and NTD joined by a flexible linker
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6
Q

describe eukaryotic RNA pol

A

5 central core proteins are HIGHLY CONSERVED (sequence of overlapping with other types)

-Pol II (makes mRNA, sm reg proteins)- Rpb 1,2,3,11,6 and couples transcription to processing of the RNA transcript

CTD (Rpb 1)- crucial to thisfunction
- repeats of Tyr-Ser-Thr-Ser-Pro-Ser
(recruits enzyme)

CAP gets added at 5’ end and mRNA direction is 5’ to 3’

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7
Q

RNA pol features

A
  • starts at promoter sequence (before gene at +1), ends at termination signal
  • proceeds in 5’-3’ direction (template is 3’-5’)
  • forms temporary DNA:RNA hybrid
  • has complete processivity (template holds on and doesn’t fall off)
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8
Q

RNA pol catalyzes _______

A

phosphodiester bond formation

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9
Q

what stabilizes phosphodiester bonding?

A

aspartic acid and magnesium

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10
Q

describe the template and coding strands

A

DNA 5’-3’ is the (+) sense, DNA coding and non-template strand

DNA 3’-5’ is the (-) antisense, DNA template strand

after template strand is transcribed, the 5’-3’ RNA transcript is made (identical to DNA coding strand but with Uracil instead of Thymine)

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11
Q
  • guides the polymerase to bind to RNA polymerase

- protein that interacts with polymerase and helps bring it to genes that need to be transcribed

A

bacterial sigma factor

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12
Q

how does bacterial promoter recognition work

A
  • sigma factor guides the pol
  • holoenzyme made and needed to get to promoter
  • sigma factor 70 interacts with Pribnow box( -10) promoter elements
  • alpha subunit of pol binds at UP element upstream of -35 element (only in some genes)
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13
Q
  • primary sigma factor in E. coli
  • essential for proteins for growth or house keeping genes
  • 4 domains
A

sigma factor 70

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14
Q

-35 promoter element bp sequence

A

TTGACA

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15
Q

Pribnow element and bp sequence

A

-10 promotor element

TATAAT

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16
Q

Domain ___ of sigma factor 70 binds 2 bases called the extended -10 region

A

3

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17
Q

what does binding to UP region of -35 promotor element promote?

A

higher rates of rRNA

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18
Q

how do eukaryotic transcription factors assemble

A

assemble at the promoter with the pol to form the pre-initiation complex

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19
Q

what is the pre-initiation complex made up of

A

TF ans pol

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20
Q

TF that stabilizes binding of TBP and TFIIB

A

TFIIA

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21
Q

TF for promoter recognition and stabilizes early transcribing complex

A

TFIIB

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22
Q

TF for promoter recognition, DNA bending, interacts with regulatory factors

A

TFIID

-key subunits: TBP, TAFs

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23
Q

TF that recruits TBIIH

A

TFIIE

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24
Q

TF that suppresses non-specific DNA binding and captures non-template strand upon melting

chaperone that brings in polymerase

A

TFIIF

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25
TF that unwinds promoter DNA and phosphorylates CTD acts as a helicase
TFIIH | - key subunits: p62, p52, XPD, XPB, MAT1, Cdk7, Cyclin H, p34, p44
26
Pol II promoter elements
core promoter - TATA box at ~-30 bases (TBP {TFIID subunit} binding site) - TBP binds at minor groove so pol can be embedded to promoter - initiator (+1) - on transcription start site - downstream element- further downstream BRE- TFIIB recognition element
27
two types of TATA-less promoters
house keeping genes and developmentally regulated genes
28
genes expressed constitutively | ex: nucleotide synthesis genes
housekeeping genes
29
describe how transcription begins and the order of TF factors
1. TFIID binds to TATA box in DNA (-35 bases) 2. TFIIA and TFIIB form complex with TFIID 3. TFIIF acts as a chaperone and brings in polymerase and binds to complex 4. TFIIE brings in TFIIH (helicase) and completes the pre-initiation complex 5. RNA pol is phosphorylated and transcription begins
30
TFIIH subunit that is mutated and repairs damaged DNA
XPB | -xerodema pigmentosum and Cockayne Syndrome
31
what is needed for initiation
mediator
32
describe a mediator
- lg protein complex (>20) required to activate transcription of many genes by pol II - interacts with CTD of pol II and regulatory proteins that bind upstream
33
mediator essential for txn of all pol II genes if mutated or missing, transcription wont occur
Med17/Srb4
34
describe initiation to elongation
- once pol is on , a closed complex formed (ready to initiate transcription) - separation of DNA strands- transcription bubble formed (14 bases)- TFIIH in euk- open complex (allow RNA formation) - template (3'-5') in active site; non-template held by rudder and zipper regions - free riboNT enter thru funnel and pairs w/ template strand - 3' OH on last NT initiates nucleophilic attack on innermost phosphate
35
successive nucleotides are added at the _____ end
3'
36
what is the first step of initiation in bacteria and eukaryotes
separation of DNA strands- transcription bubble formed (14 bases)- TFIIH in euk- open complex (allow RNA formation)
37
what happens when the innermost phosphate is attacked
breaking of the bonds gives energy to move along in elongation
38
what ions are in the active site
two Mg 2+ ions
39
what is occurring at the active site
- two Mg2+ ions activate 3'OH for attack and stabilizing the negative charge of the oxygens leaving - one is bound to pol and the other is with the incoming nucleotide
40
RNA pol fails to make a full length RNA on the first attempt; 2-9 nucleotides in length released
abortive initiation
41
abortive initiation factors that extend in the active site and impede
sigma factor (loop3.2) or TFIIB (B finger loop)
42
displacement of the protein loop is thought to help the polymerase break away from the promoter
promoter clearance
43
what happens to the RNA pol during promoter clearance
RNA pol undergoes conformational change (in euk and prok) that associates it very stably with DNA and loosens grip on initiation factors
44
difference in eukaryotic and prokaryotic promoter clearance
eukaryotic -pol is phosphorylated (TFIIH) as it converts to the elongating complex prokaryotic - sigma factor and initiation factors are then released - as pol unwinds DNA, it re-winds behinds it causing (+) supercoiling up front and (-) supercoiling from re-winding
45
bacteria vs eukaryotic transcription
bacteria - closed complex: holoenzyme + promoter - open complex: DNA melting, NO ATP - abortive transcription: sigma factor (loop 3.2) - productive initiation: transcribe past +9 and sigma conformational change (sigma left behind) - elongation eukaryotic - pre-initiation complex assembly (TF and pol) - PIC activation: melting DNA, needs ATP - abortive initiation: TFIIB (B finger loop) - productive initiation: CTD phosphorylated and promoter clearance - elongation
46
why is processivity high during transcription elongation
bc the pol is closed around the DNA like a set of jaws
47
what favors the polymerization process during transcription elongation
cleavage of incoming NT and the subsequent cleavage of the pyrophosphate
48
how many NT are added during transcription elongation
15-20 NT added per sec
49
the transcription bubble during transcription elongation contains how many bases
10-14 bases maintained
50
if DNA secondary structure is too bulky what can happen with the pol
it can fall off
51
during elongation, why can transcription pause
- hairpin structures | - weak DNA-RNA hybrid in the bubble bc of AU rich sequences
52
translocation with the ELL genes and pTEFb genes
AML
53
involved in promoter proximal pausing (-35-50 bases in)
NELF and DSIF
54
promoter proximal pausing is for
5' capping
55
eukaryotic elongation factor that disrupts interaction with TBP and TFIIB
ELL
56
eukaryotic elongation factor that phosphorylates CTD of Rpb1 subunit of RNA pol II
p-TEFb
57
negative elongation factor promotes pausing
NELF/DSIF
58
which pol makes mRNA called pre-mRNA
RNA pol II
59
what is coupled with mRNA processing in eukaryotes
mRNA processing is coupled with transcriptional elongation
60
what recruits capping enzymes in elongation
the fifth serine in the CTD heptad repeat
61
where does pTEFb phosphorylate on the CTD
the 2nd serine in CTD
62
what happens when the elongation complex is paused
complex can backtrack
63
factors that promote hydrolysis of the phosphodiester bonds
transcription cleavage factors
64
how can eukaryotic elongation have a role in error correction
if mismatched bases are incorporated, theDNA-RNA duplex could be distorted and cause stalling of RNA pol
65
describe elongation in eukaryotes
- TFIIH phosphorylates serine 5 on CTD of pol - negative elongation factors(ELL/p-TEFb) bind pol and arrest transcription - capping enzyme modifies 5' end of transcript - pol stalled - backtracking - transcript cleavage - transcription elongation
66
examples of transcript cleavage factors in E. coli
GreH and GreB
67
transcript cleavage factor in eukaryotes
TFIIS
68
transcript cleavage factors bind within the ______ region of RNA Pol
funnel
69
when RNA pol backtracks, which end of the RNA extrudes through the funnel
3'
70
describe nucleosome blocking elongation
histone chaperones disassemble and reassemble and added back to template once it has been read
71
histone chaperone that binds H2A-H2B dimers
FACT (facilitates chromatin transcription)
72
histone chaperone that binds H3-H4 tetramers
Asf1
73
histone chaperone that binds H3-H4 and H2A-H2B
Spt6
74
the transcription bubble moves along DNA, continually unwinding the duplex, what can lead it lead to
changes in supercoiling
75
describe the supercoiling change in elongation
``` before pol (-) supercoiling after pol (+) supercoiling ```
76
relieves tension from changes in supercoiling
topoisomerases
77
what can changes in supercoiling cause
could cause the RNA pol to stall
78
in E. coli, what removes the (+) and (-) supercoils
(+) supercoils- DNA gyrase | (-) supercoils- DNA topoisomerase 1
79
what happens when tension from supercoiling is not released
pol can stall and if it doesn't get fixed, then it will fall off
80
specific sites on template stand in bacteria
terminators
81
two types of termination in bacteria
intrinsic terminators | rho(p) dependent terminators
82
hydrolyzes ATP in bacteria termination
rho-hexameric ATPase
83
termination of RNA pol II genes is coupled to processing of the ______
3' end of mRNA
84
what do most eukaryotic mRNAs have
polyA tail
85
describe eukaryotic termination by pol II
RNA pol II continues to translate after the polyadenylation signal, but the mRNA is cleaved at the poly(A) signal and the 3' end of the mRNA is processed
86
two main models of transcription termination by RNA pollII
allosteric and torpedo
87
describe allosteric model transcription termination
- RNA pol II transcribes thru the poly(A) and 3' processing signals - RNA processing proteins associate w/ the processing signals and CTD - cleavage/recognition of processing proteins causes confirmation changes that lead to disassociate of pol II form DNA
88
termination is linked to
degradation of cleaved RNA fragment
89
describe torpedo model transcription termination
- after cleavage, RNA downstream of poly(A) tail site is digested by a 5' to 3' endonuclease (RAT1) - endonuclease is the torpedo: continues to degrade the nascent RNA until it runs into RNA pol - this disrupts polymerization and causes the enzyme to disassociate from the describe allosteric model transcription termination

Molecular Biology (4 decks)

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