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MLSCI 480 > Eukaryotic Transcription > Flashcards

Eukaryotic Transcription Flashcards

1
Q

T o F. Not all genes code for proteins, some RNA products are functional

A

T! rRNA and tRNA

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2
Q

RNAP I

A

RNA polymerase I
- transcription of rRNA precursors

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3
Q

RNAP II

A

RNA Pol II
- transcription of mRNA precursors

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4
Q

RNAP III

A

RNA Pol III
- transcription of 5S rRNA, tRNA, and other small RNA precursors

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5
Q

largest subunit of RNAP II

A

Rpb1

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6
Q

phases of transcription

A

assembly
initiation
elongation
termination

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7
Q

cofactors of RNAP II

A

Mg 2+
Zn 2+

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8
Q

transcription factors required to initiate transcription by RNAP II

A

6 general transcription factors
- required for synthesis of all mRNAs
- names begin with TFII; (TF-transcription factor; II-RNAPII)

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9
Q

this binding protein is the first protein to bind to a promoter in initiation

A

TATA binding protein
- part of TFIID along with TBP-associated factors or TAFs
- binding distors the DNA, partially unwinding the duplex
- hydrogen bonding and vDW interactions
- Pol II by itself is unable to bind to a promoter

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10
Q

TFIIH

A

helicase
phosphorylation of CTD

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11
Q

T or F. Eukaryotes lack precise transcription termination sites

A

T!
no identified transcriptional termination sie in eukaryotes

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12
Q

RNA polymerase undergoes selective _____________.

A

inhibition
- rifampicin
- actinomycin D
- alpha-amanitin

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13
Q

Rifampicin

A
  • antibiotic
  • inhibits bacterial B subunit of RNA pol
  • prevents promoter clearance
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14
Q

actinomycin D

A
  • anti-cancer agent
  • inhibits bacterial and eukaryotic RNA pol
  • intercalates into dsDNA preventing movement of RNA and RNA pol along template
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15
Q

alpha-amanitin

A
  • inhibits eukaryotic RNA pol
  • blocks pol II (at higher concentrations, pol III too)
  • does not block bacterial pol or Pol I
  • from poisonous death cap mushrooms
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16
Q

function of 5’ cap

A
  • protects mRNA from ribonucleases (resistant to 5” exonucleases)
  • binds to specific cap-binding complex
  • participates in binding to ribosome to initiate translation
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17
Q

T or F. RNA is synthesized from the coding or sense strand

A

F! RNA comes from the antisense strand

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18
Q

T or F. The coding strand sequence matches the sequence of RNA

A

T! It is used as a reference for the sequence

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19
Q

Additional RNAPs will exist in organelles like ___________ and ______________.

A

mitochondria and chloroplasts
- some subunits are shared b/w I,II, and III
> omega homolog = identical in all 3
> alpha homolog = identical in I and III

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20
Q

carboxy-terminal domain with consensus sequence

A

lots of Ser
- what is phosphorylated
- serines susceptible to covalent modification by phosphorylation (CTD kinase, CTD phosphatase)
- elongation/initiation sensitive to phosphorylation state of CTD

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21
Q

RNAP II active site

A

clamp portion of Rpb2 locks over bound DNA to increase processivity

contacts between RNAP and hybrid duplex are primarily to backbone

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22
Q

what are eukaryotic promoters?

A
  • promotes process; knows where to start!
  • each polymerase has its own associated promoter types
  • accessory proteins identify promoters and recruit RNAP to transcription start site
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23
Q

upstream is …

A

negative (left)
where initiation happens

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24
Q

elements in a pol II promoter

A

TFIIB
TFIID (TATA binding protein)
MTE/PDE elements = used in absence of TATA box

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25
Q

describe the formation of the pre-initiation complex

A
  1. TBP component of TFIID binds to TATA box of promoter
  2. TFIIA & TFIIB bind
  3. TFIIF binds to RNAP II and escorts it to the complex
  4. TFIIE and TFIIH are sequentially recruited, thereby completing the PIC
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26
Q

TFIIB interacts with both RNA Pol II via ________ domain and TBP via _______ domain

A

N-terminal; C-terminal

27
Q

T or F. One RNAP = one mRNA

A

T!

28
Q

Transcription is iniated after…

A

pre-initation complex is assembled
and then Pol II CTD is phosphorylated

29
Q

TFIIH

A
  • essential for nucleotide-excision repair complex
  • site of DNA lesion = Pol II halts, TFIIH interacts with lesion => recruits entire nucleotide-excision repair complex
  • result = repair of damaged DNA more efficient on actively transcribed genes and template stand is repaired more efficiently than non-template strand
30
Q

T or F. There is no transcript stop signal in eukaryotes

A

T! unlike bacteria; but there is a poly-A tail that exists for translation to occur

31
Q

T or F. Prokaryotic mRNA protein coding is not as tightly processed as eukaryotic coding

A

T!
not usually modified (translation initiates before synthesis is complete in prokaryotes)

32
Q

eukaryotic mRNA primary transcripts are extensively modified here

A

nucleus

33
Q

T or F. tRNA and rRNA are modified in both prokaryotes and eukaryotes

A

T

34
Q

this removes introns from primary transcripts

A

splicing

35
Q

where does eukaryotic transcription occur?

A

nucleus
primary transcripts undergo extensive modification prior to going to cytosol (translation)

36
Q

coding and non-coding eukaryotic transcripts

A

coding = exons
non = intorns

37
Q

function of 5’ cap

A
  • protects mrNA from ribonucleases (resistant to 5’ exonucleases)
  • binds to specific cap-binding complex
  • participates in binding to ribosome to initiate translation
38
Q

describe the 5’ cap

A

7-methyguanosine attached to 5’ nucleotide via triphosphate linkage

2-hydroxyls of first and second nucleotides often methylated

capping enzymes bound to RNAPII’s phosphorylated CTD

added early in transcription (after first 20-30 nucleotides)

39
Q

process of added 5’ cap

A

removal of 5’ phosphatase - hydrolysis

GTP addiiton

methylation of G- generating the mature cap

40
Q

synthesis of the 5’ cap is carried out by enzymes tethered to the _________ terminal domain of POLII

A

carboxyl
- enzymes synthesize cap are tethered to RNAPOLII CTD
-cap remains tethered to CTD by camp-binding complex/CBCC
> protects RNA from degradation
> involved in transport

41
Q

describe the eukaryotic poly A tail

A

80-250 residues at 3’ end of primary transcript

protein binding site (polA binding proteins)

protects RNA from degradation (without polY, longevity of RNa greatly reduced)

poly A not encoded in the genome
- added in a multi step process
> recognition of sequence
> cleavage
> addition of A

42
Q

process of binding polyA tail

A
  • conserved AAUAAA sequence is 10-30 nts upstream of polyA
  • transcript extend beyond where polyA is added
  • enzyme complex with endonuclease (polyadenylate polymerase) associated with CTD and binds AAUAAA

transcript cleaved by endonuclease

A residues are added to free 3’-OH by polyadenylate polymerase

  • no template required
43
Q

expressed regions

A

exon

44
Q

intervening/intragenic regions

A

introns

45
Q

T or F. Introns occur in majority of vertebrate genes

A

T!
- only known vertebrate structural genes that lack introns = histone and interferon genes

46
Q

size of introns vs exons

A

exons = <1000 bp

introns = 50- 20 000 bp

NOTE: human genes have more DNA devoted to introns than exons
~80% of typical vertebrae structural gene is composed of introns

47
Q

four general classes of introns

A

group I and II are self-splicing = auto-catalytic
> no proteins involved required in vitro

spliceosomal introns = nuclear mRNA primary transcripts and splicing is mediated via a large protein complex

unnamed class of introns = certain tRNAs; require ATP and an endonuclease

48
Q

Group I vs II introns

A

I = nucleus, mitochondria, chloroplast genes (rRNA, mRNA, tRNA) in diverse eukaryotes not vertebrates, and in some bacteria

II = primary transcripts of organelles (mitochondria and chloroplasts) of fungi, algae, and plants

both do not require ATP; transesterification

49
Q

largest class of introns

A

spliceosomal introns

50
Q

where are spliceosomal introns found?

A

nuclear mRNA primary transcripts
- heterogenous nuclear RNA and pre-mRNA

51
Q

mechanism of spliceosomal introns

A

lariat-forming mechanism (similar to group II introns)
- requires large splicing complex - spliceosome

made up of RNA-protein complexes (snRNPs - small nuclear ribonucleo proteins)

5 snRNAs = U1, U2, U4, U5, U6
~50 proteins
comparable in size and complexity to the ribosome

energy spent!

52
Q

what do spliceosomes do?

A

recognize and bring two exons together
- 5’ of U1-snRNA = partially complementary to consensus sequence of 5’ splice junctions
- U1-snRNP recognizes the 5’ splice junction

spliceosome is highly dynamic = various components associating and dissociating during specific stages of splicing process

53
Q

spliceosome complexes remove _____ in nuclear ______ primary transcripts

A

introns; mRNA

54
Q

how do spliceosome complexes remove introns in mRNA primary transcripts?

A
  • U1, U2 recognize exon intron junction
    > dinucleotide AG , GU flank intron (consensus)

> U1 complementary sequence that pairs to consensus 5’ splice site
U2 = intron sequence (internal) that contains A residue that will perform nucleophilic attack

  • base pairing forms bulge around A = activates it
    > conformation around A is a duplex structure different from other regions
  • ATP consumed during assembly phase (not during splicing)
  • transesterification => lariat formation w joining of exon terminal nucleotides
55
Q

T or F. Complexity of organism does not correlate with number of genes

A

T

56
Q

Poly(A) site choice generates __________ in variable domains of immunoglobulin heavy chains

A

diversity

  • polyA affactes stability
57
Q

what does alternative splicing produce in fruit fly development?

A

3 different forms of myosin heavy chain transcript at different stages of development

58
Q

T or F. rRNAs and tRNAs also undergo processing

A

T!

  • made from longer precursors; so must be smaller
  • contain modified nucleosides; must be inncorporated
  • tRNAs (40-50 in most cells)
    > enzymatic removal of 5’ & 3’ ends of longer precursor
    > 3’-terminal CCA that attaches to amino acid for protein synthesis is added by tRNA nucleotidyltransferase
    > base modification (methylation, deamination, reduction)
59
Q

ribozymes

A
  • self-splicing group I and II introns, RNAse P
  • catalyze 2 rxns = transesterification and hydrolysis of phophodiester bonds
  • subsrates often RNA, in which case base-pairing can align the substrate for rxn
  • 3D structure important for function and therefore are inactivated by denaturation = just like proteins
60
Q

RNA stability in eukaryotic cell

A

average = 3h with 10x turnover within a cell generation
- may be seconds, minutes or stable over many generations
- 5’ and polyadenylation IMPORTANT

61
Q

what degrades RNAs?

A

ribonucleases

62
Q

RNA degradation in lower eukaryotes

A

shortening of polyA tail, decapping, mRNA degradation in 5’->3’ direction

63
Q

RNA degradation in higher eukaryotes

A

have 3’->5’ degradative pathway

MLSCI 480 (16 decks)

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