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Mcb1 > RNA Metabolism > Flashcards

RNA Metabolism Flashcards

1
Q

Is the coding strand identical or complementary to the mRNA (except for T/u)?

A

The coding strand is identical

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2
Q

Is the DNA template strand complementary or identical to the coding strand?

A

DNA template strand is complementary and antiparallel to the mRNA

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3
Q

In what direction is mRNA synthesized?

A

5’ to 3’ from the template strand

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4
Q

In what direction do ribosomes translate mRNA?

A

In the 5’ to 3’ direction, synthesis goals the protein from the amino terminus to the carbonyl terminus

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5
Q

When the template strand is copied into RNA, what strand is it on the DNA helix?

A

The 3’ to 5’ strand in the DNA double helix (the “bottom strand” in written DNA)

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6
Q

What strand is the coding strand?

A

The non-template strand (the “top strand” in written DNA)

Can be read as the RNA strand provided all T’s are read as U’s

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7
Q

How many RNA polymerase are found in bacteria?

A

Only 1 type

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8
Q

Does RNA polymerase have proofreading ability?

A

Nope, it makes errors at a rate of 1 in 10k bases

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9
Q

When can RNA polymerase start transcription?

A

At a particular DNA sequence in front of the gene (promoter)

It does not require a primer

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10
Q

Where does RNA polymerase bind when the promoter or promoter site is found?

A

From 70 bp’s before the transcription start site to 30 bp after the transcription start site

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11
Q

What is the sigma factor?

A

The part of the promoter site which binds to RNA polymerase.

  • a detachable subunit of RNA polymerase and guides it to the promoter
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12
Q

What is the combination of the core enzyme (RNA polymerase) and the sigma factor called?

A

The holoenzyme

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13
Q

What drives the reaction of RNA polymerase?

A

Hydrolysis of pyro-phosphate drives the reaction

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14
Q

Where does RNA polymerase attack in order to grow an RNA chain?

A

RNA polymerase catalyzes an attack on the 3’ OH of the nucleotide at the end of the growing strand, using the 5’ alpha-phosphate of the incoming NTP

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15
Q

How do you denote the DNA sequence of a transcribed DNA?

A

The first nucleotide (start site) of a transcribed DNA sequence (coding strand) is denoted as +1 and the second one as +2 etc

The nucleotide preceding the start site is -1

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16
Q

What is the pribnow box?

A

The -10 region in a bacterial promoter sequence

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17
Q

What are the two conserved promoter sequences found in bacteria?

A
  • 35 region

- 10 region, also called the pribnow box

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18
Q

How does RNA polymerase determine orientation of promoter sequence?

A

An RNA that moves from left to right makes RNA by using the bottom strand as a template

An RNA polymerase that moves from right to left makes RNA by using the top strand as template

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19
Q

what are the 3 factors important for promoter strength?

A
  1. The consensus sequences
  2. The spacing between the consensus sequence
  3. The distance of the consensus sequences from the transcription start site
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20
Q

What can increase efficiency of the promoters?

A

Negative supercooling by topoisomeases can increase efficiency of promoters

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21
Q

After the promotor is cleared and RNA polymerase attaches what happens?

A

A major conformational change that cannot revert

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22
Q

How many Bp remain unwound during transcription?

A

15-17 bp remain unwound during transcription (= transcription bubble) and RNA/DNA hybrid stays limited to 7bp

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23
Q

What are the two termination of bacterial transcription?

A
  1. Rho factor

2. Rho factor-independent

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24
Q

What is the Rho factor?

A

An ATP-dependent helicase which can unwind RNA/DNA hybrids

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25
Q

Where does the Rho factor attach to and what does this do.

A

The Rho factor bind to a CA-rich region in the nascent RNA chain some distance upstream of the termination site and then catches up with the RNA polymerase (transcription bubble) and dissolves the mRNA-DNA-polymerase complex (RNA strand).

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26
Q

What is the Rho factor independent termination of bacterial transcription?

A

Terminated by a combined action of weak U-A bonding in the RNA/DNA hybrid and strong G-C bonding in the nascent RNA

I.e the hairpin of the nascent RNA; the RNA fold on itself

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27
Q

What does Rifampin do?

A

Binds to RNA polymerase and changes its conformation so that it cannot initiate RNA synthesis.

RNA polymerase from eukaryotes cells does not bind rifampin, and RNA synthesis is unaffected

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28
Q

What is actinomycin?

A

An antibiotic that binds strongly to double stranded DNA (prevents it from being an effective template for RNA synthesis)

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29
Q

What happens when you have high concentrations of actinomycin?

A

Inhibits DNA replication

It’s specificity for double stranded DNA can be explained by intercalation

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30
Q

What doe the antibiotic rifampin inhibit?

A

Initiation of transcription by binding to the RNA polymerase

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31
Q

What is the location of RNA polymerase in I, II, III?

A

I: Nucleolus

II: Nucleoplasm

III: Nuceloplasm

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32
Q

What are the cellular transcripts of eukaryotic RNA polymerase I, II, III?

A

I: 18s, 5.8s and 28s rRNA

II: mRNA precursors, snRNA and miRNA

III: tRNA and 5S rRNA

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33
Q

What are the effects of alpha-amanitin (from mushrooms) on the Eukaryotic RNA polymerase?

A

I: insensitive

II: strongly inhibited

III: inhibited by high concentrations

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34
Q

What part of the Eu RNA polymerase differ?

A

The C-terminal part, which is substantially longer for RNA pol II

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35
Q

What is alpha-amanitin?

A

A poison found in mushrooms

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36
Q

How many subunits does the bacterial RNA polymerase have?

A

5

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37
Q

What does Eukaryotic RNA polymerase II require for it to begin transcription?

A

“General transcription factors” (TFIIX)

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38
Q

What is DPE?

What’s the consensus sequence and transcription factor?

A

Downstream promoter element

Consensus: A/G G A/T C G T G

General transcription factor: TFIID

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39
Q

What does TFIID do?

A

TBP subunit (1): recognizes TATA box

TAF subunits (~11): recognizes other DNA sequences near the transcription start point; regulates DNA-binding by TBP

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40
Q

What does TFIIB do?

A

Recognizes BRE element in promoters

Accurately positions RNA polymerase at the start site of transcription

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41
Q

What does TFIIF do?

A

3 subunits

Stabilizes RNA polymerase interaction with TBP and TFIIB; helps attract TFIIE and TFIIH

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42
Q

What does TFIIE do?

How many subunits

A

2

attracts and regulates TFIIH

43
Q

What does TFIIH do? How many subunits?

A

9 subunits

Unwinds DNA at the transcription start point

Phosphorylates Ser5 of the RNA polymerase CTD

Releases RNA polymerase from the promoter

44
Q

What is CTD?

A

Carboxy-terminal domain

45
Q

When are most of the transcription factors released by after the complex starts.

A

60-70 nucleotides (nt)

46
Q

How is RNA polymerase recycled in eukaryotes?

A

Dephosphorylation

47
Q

What does RNA polymerase require for removal of the positive supercoils in front of it?

A

Topoisomerase activity

48
Q

Why does eukaryotic RNA polymerase require elongation factors?

A

To displace nucleosomes (with ATP-dependent remodeling complexes)

Transciently dislodging histone dimers

49
Q

What is the corresponding segment of DNA from which a primary transcript is transcribed called?

A

Transcription unit

50
Q

What does RNA processing require in eukaryotes?

A

A variety of small RNA’s of 90-300 nt (the small nuclear RNA’s, snRNA’s) and associated proteins, in addition to the enzymes involved in capping and polyadenylation

51
Q

Does mRNA in bacteria undergo modification? Why or why not?

A

It undergoes little or no modification because it is being translated while being transcribed

52
Q

What does guanylyltransferase do?

A

Caps the nascent mRNA chain

Note: subsequent methylation at N7 is various out in the cytoskeleton by the enzyme guanine-7-methyltransferase

53
Q

What does guanine-7-methyltransferase do?

A

Methylated the mRNA at N7 in the cytosol

54
Q

What is the purpose of capping?

A

Capping protects the mRNA from degradation and is needed for export out of the nucleus and for assembly of a translation complex

55
Q

Are tRNAs and rRNAs capped?

A

They are not capped

56
Q

What is AAUAAA?

A

The polyadenylation signal sequence which is located in the 3’ UTR and the nascent mRNA is cleaved within 10-40 bases downstream of it by a specific endonuclease.

57
Q

What cleaves the mRNA after the poly-A tail?

A

Endonuclease

58
Q

What carries out the polyadenylation reaction ?

A

Polyadenylate polymerase

59
Q

What is the purpose of the Poly-A tail?

A

To stabilize the mRNA, is is needed for the nuclear export of the mRNA, and for assembly of the translation complex

60
Q

What happens to the polyA tail in the cytoskeleton?

A

It gradually shortens

61
Q

Is the Poly-A tail encoded for in the DNA? (25-200 A’s)

A

No

62
Q

What happens after the poly-A tail is added?

A

Splicing

63
Q

What does splicing do?

A

Puts together the final sequence (exons)

64
Q

What enzyme carries out molecular assembly via 2 transesterications,

A

Splicesome

65
Q

By how much can introns vary by??

A

Between 50 and 10k nucleotides

66
Q

What do large insertions and deletions do to the splicing of an mRNA?

A

Large deletions or insertions do not affect splicing of the intron in most cases

67
Q

All eukaryotic introns start with what and end with?

A

Start with GU

End with AG

68
Q

What is the 5’ and 3’ splice sites called?

A

5’ = donor site

3’ = acceptor site

69
Q

What are the two transesterifications in splicing?

A
  1. Nucleophilic attack of a branch site A on the 5’- phosphate group of the intron
  2. Nucleophilic attack of the now free 3’-OH group of upstream exon on the 5’-phosphate of the downstream exon
70
Q

What part of the spliceosome assembly recognizes the 5’ splice site (donor site)

A

The U1 snRNP

71
Q

What does the U2 snRNP of the spliceosome do?

A

Bind to the branch site (A site) recognized by U1

72
Q

When U2 binds to the branch site recognized by U1, what happens?

A

A preasssemblex complex of U4-U5-U6 joins the others to form a complete spliceosome (inactive)

73
Q

After the (inactive) spliceosome is completed, what does U5 do next?

A

U5 interacts with exon sequences on the 5’ splice site and the 3’ splice site and pulls the splice sites together

74
Q

After U5 brings the 5’ and 3’ prime ends together, what happens next?

A

U6 detaches from U4 and binds to U2

Note: U1 leaves with U4

75
Q

What do U6 and U2 snRNP function as? ( This occurs after U6 detaches from U4)

A

U6 and U2 snRNP function as ribozymes catalyzing both transesterications

76
Q

How is the branch site A activated?

A

The RNA duplex formed with U2 forces the A nucleotide out of the RNA duplex making the 2’OH reactive

77
Q

What is the UTR and what’s it do?

A

The untranslated regions (UTRs) can be much longer than the coding region and contains regulatory elements important for RNA stability and translation efficiency.

Note: the 5’-UTR consist of (part of) the first exon(s) and the 3’-UTR of (part of) the last exon(s)

78
Q

How do variant proteins arise?

A

There are 3 sources for variant mRNA:
1. Alternative start sites (multiple promotors)

  1. Alternative splicing
  2. Alternative termination sites
79
Q

The half life of bacterial mRNA are unstable with what half life?

What benefit does this offer?

A

A few minute so that bacteria can rapidly respond to changes in the environment

80
Q

What does the intracellular mRNA concentration depend on?

A

Synthesis and degradation within the cell

81
Q

What is the half life of mRNA in eukaryotes?

A

A few minutes for proteins involved in the cell cycle to 24 hrs for globin mRNA in erythroid cells (erythrocyte)

82
Q

What is the mechanism for bacterial mRNA degradation?

A

An endoribonuclease makes a few cuts in the mRNA followed by 3’ to 5’ exoribonuclease action

83
Q

What is the mechanism for mRNA degradation in Eukaryotic cells?

A

Shortening of poly A tail acts as a timer counting down the lifetime of an mRNA

Degradation occurs in specialized regions in the cytoplasm calles exosomes

84
Q

What is an exosome?

A

A specialized region in the cytoplasm where degradation of mRNA occurs

85
Q

What happens when the polyA tail becomes short than ~25 A’s?

A

The mRNA is rapidly degraded by either recapping followed by 5’ to 3’ degradation or 3’ to 5’ degradation

86
Q

How many ribosomal RNAs are there in eukaryotes and bacteria? (rRNA)

A

4 in Eu

3 in bacteria

87
Q

Why are rRNAs not frequently broken down?

A

Bases are frequently modified: methylation of bases and/or of the ribose unit as well as isomeric action of uridine to pseudouridine

88
Q

What rRNA does not cluster?

A

5S rRNA

89
Q

Where does transcription of rRNA genes and processing of eukaryotic rRNA take place?

A

In the nucleolus

90
Q

What is the Svedberg number?

A

A number given to rRNA based on old techniques used to determine size

91
Q

How are tRNAs made?

A

Derived from longer RNA precursors by enzymatic removal of nucleotides at both the 5’ and 3’ end

92
Q

Both bacterial and eukaryotic tRNAs have a CCA sequence at the 3’ end. What is different about the eukaryotic tRNA?

A

The CCA in eukaryotes sequence is added posttranscriptionally

93
Q

What s the anticodon for recognizing multiple codons for tRNA?

A

Inosine

94
Q

How many distinct tRNAs do cell have?

A

40-50

Note: cancer cells have about 80-90

95
Q

What do some eukaryotic tRNs contain?

A

An intron that has to be spliced out

96
Q

What transcribe micro RNA?

A

miRNA is transcribed by RNA pol II that are capped, polyadenylated, processed in the nucleus, and further processed by Dicer in the cytoplasm

97
Q

What is Dicer?

A

The enzyme that proceses ~75 nucleotides~ long genome-encoded RNAs with profound hairpin structure into double stranded silencing- RNAs (siRNAs) ! 23 nt in length

98
Q

What is the RISC complex involved in?

A

Regulation of the stability and translatability of mRNAs

99
Q

What is silencing-RNAs (siRNAs) used for?

A

It is incorporated into the RNA-Induced Silencing Complex (RISC)

100
Q

Are all miRNAs forming siRNAs?

A

No, some seem to enhance/stabilize

101
Q

What are the guide/scanning RNAs?

A
  1. CRISPR-RNA

2. Scan-RNA

102
Q

What are some classes of long non coding RNA? (Non coding means it doesn’t make a protein)

A
  1. Telomerase RNA
  2. Transcriptional enchanter element RNAs
  3. Multifunctional IncRNAs
103
Q

What are the three sources for variant mRNA?

A
  1. Alternate start site
  2. Alternate splicing
  3. Alternate termination site

Mcb1 (13 decks)

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