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Nucleic Acid Biochem II > 20-22 Transcription and its control > Flashcards

20-22 Transcription and its control Flashcards

515-540 734-742

1
Q

What are three similarities between DNA replication and transcription?

A
  • Incorporation of nucleoside tripohsphates
  • DNA template directed
  • Template-directed extension of nucleic acid chains in 5’ to 3’ direction
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2
Q

What are 4 differences between DNA replication and transcription?

A
  • Only one DNA strand is trasncribed
  • RNA primers not required for transcription
  • All of the DNA in a cell is replicated by DNA polymerase, but only a fraction of the genome is transcribed and only a fraction of the genes are transcribed at any one time
  • Different enzymes involved, with very different characteristics
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3
Q

In prokaryotic transcription, the three types of RNAs are transcribed by what enzyme(s)?

A

All three are transcribed by RNA polymerase

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4
Q

What are the three broad steps of prokaryotic transcription?

A
  • Initiation
  • Elongation
  • Termination
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5
Q

What are the two steps of initiation in prokaryotic transcription?

A
  • Binding of RNA polymerase to promoter to form closed complex (binding)
  • DNA unwound at around +2 or +3 to form open complex (initiation) and alpha subunit of RNA polymerase leaves to start elongation
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6
Q

During transcription, is there a lot of protein contact?

A

Yes, it is extensive with RNA, DNA and RNA/DNA together

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7
Q

How many bp of DNA are unwound within the prokaryotic transcription bubble?

A

17 bp

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8
Q

Prokaryotic RNA polymerase can pause and translocate backwards to?

A

Cleave the 3’ end of nascent RNA

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9
Q

What are prokaryotic promoter regions?

A

Conserved sequences that are non-template (aka sense) recognized by prokaryotic RNA polymerase.

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10
Q

At what region of prokaryotic promoter is the Pribnow box and Initiation site?

A

Pribnow box = -10 region

Initiation site = +1 region

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11
Q

What are the consensus sequences of a promoter region in prokaryotes? (3) What space between two of these is critical for RNA polymerase binding?

A
  • -35 region
  • -10 region
  • Initiation site (+1)

Space between -35 region and -10 region critical for RNA polymerase binding

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12
Q

What is the consequence of prokaryote promoter mutations?

A

Either up-promoter mutation or down-promoter mutation, which increases or decreases promoter strength respectively.

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13
Q

How does factor-independent termination in prokaryotes work?

A

Factor-independent termination

A G/C rich hairpin palindrome precedes a A-rich segment on template strand. The hairpin pauses RNA polymerase and A-U base pairs disconnect, causing transcript to detach from template

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14
Q

How does factor dependent transcription termination work in prokaryotes? What is the factor called?

A

Rho factor-dependent termination

ρ (Rho) factor is recognizes CA rich regions (rut sites) near the 3’ end of the transcript where RNA pol has paused. Rho moves towards the 3’ end of teh transcript by unwinding the RNA-DNA duplex and pulling it away from the RNA polymerase.

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15
Q

Which type of prokaryotic transcription termination ATP-dependent?

A

Rho factor-dependent termination

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16
Q

What is the structure of the Rho termination factor in prokaryotes?

A

An RNA-DNA helicase with nucleoside triphosphatase activity that is activated upon binding to polynucleotides

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17
Q

What are two methods used for mapping prokaryotic transcriptional start points?

A
  • Primer extension method

- Footprinting

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18
Q

What are the steps of the primer extension method for mapping prokaryotic transcriptional start points?

A
  • A end-label restriction fragment (or primer) is used internal to the promoter
  • This fragment is hybridized downstream from promoter and radioactively labeled at 5’ ends
  • Reverse transcriptase extends 3’ end of DNA primer until it reaches the 5’ end of the template RNA
  • The extended product is run on sequencing gel and autoradiography reveals length of the fragment which can be used to determine the start point of transcription
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19
Q

What are the steps of the footprinting method for mapping prokaryotic transcriptional start points?

A

DNA footprinting is a method of investigating the sequence specificity of DNA-binding proteins in vitro

  • PCR amplifies region of DNA with potential protein binding site
  • Protein of interest added to DNA solution, another mixture with protein is isolated for comparison
  • Add nucleases, which will not cut DNA protected by bound protein
  • Run both samples on gel electrophoresis. A ladder will be made with both samples, though the portion of DNA protected with protein will not show bands (there will be a gap in the ladder)
  • This can be used to determine where RNA polymerase binds to DNA (promoter region)
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20
Q

What are the additional proteins needed to recognize promoter and initiate transcription called?

A

Transcription factors

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21
Q

What are transcription factors?

A

Proteins with DNA binding domains and one or more regulatory domains used to interact with RNA polymerases and other proteins involved in transcription

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22
Q

Are transcription factors specific to specific RNA polymerase complexes?

A

Some of them are complex-specific, some of them are common to all three RNAP complexes.

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23
Q

What eukaryotic RNA polymerase synthesizes pre-rRNA (except 5S)?

A

RNA polymerase I

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24
Q

What eukaryotic RNA polymerase synthesizes pre-mRNA and some small nuclear RNAs?

A

RNA polymerase II

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25
Q

What eukaryotic RNA polymerase synthesizes pre-tRNA, 5S rRNA and other small RNAs?

A

RNA polymerase III

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26
Q

Where do RNA polymerase I, II and III work?

A

II and III work in the nucleus, I works in the nucleolus.

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27
Q

What are three similarities between eukaryotic and prokaryotic RNA polymerase II elongation complexes?

A
  • Issues with supercoiling
  • Similar size and structure of the transcription bubble
  • Protein-RNA and protein-DNA as well as protein-protein interaction
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28
Q

NTPs enter the RNA polymerase II elongation complex at which part of it?

A

The funnel, adjacent to the active site

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29
Q

What cofactor must be at the active site of RNA polymerase for transcription to occur?

A

Mg 2+

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30
Q

Is transcribed DNA upstream or downstream to the RNA polymerase elongation complex?

A

Upstream

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31
Q

Is entering DNA upstream or downstream to the RNA polymerase elongation complex?

A

Downstream

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32
Q

What are two proteins (or types of proteins) essential for RNA polymerase I?

A
  • Transcription factors

- TATA binding protein (TBP)

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33
Q

What are the ribosomal subunits that result from RNA polymerase I transcription in eukaryotic cells?

A
  • 40S

- 60S

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34
Q

What pre-rRNA does RNA polymerase I transcription yield in mammalian eukaryotic cells?

A

mammalian 45S pre-rRNA

35
Q

What are the rRNAs that result from multistep rRNA processing of 45S pre-rRNA made from RNA polymerase I?

A
  • 18S
  • 28S
  • 5.8S
36
Q

What two processes happen in the nucleolus?

A
  • rRNA transcription by RNA polymerase I

- RIbosomal biogenesis

37
Q

What types of molecules does the nucleolus contain?

A
  • DNA
  • RNA
  • Protein
38
Q

What does RNA pol III transcribe in eukaryotic cells?

A
  • pre-tRNA
  • 5S rRNA
  • Other small RNA genes (multi-copy genes, although typically not arranged in tandem repeats)
39
Q

How many subunits does eukaryotic RNA polymerase I have?

A

13

40
Q

How many subunits does eukaryotic RNA polymerase III have?

A

14

41
Q

What are the three protein factor required for RNA pol III transcription? What do these do?

A
  • TFIIA (5S rRNA)
  • TFIIB (5S rRNA and pre tRNA)
  • TFIIC (5S rRNA and tRNA genes)
42
Q

Which eukaryotic protein factor of RNA polymerase III has a zinc finger for DNA-protein binding?

A

TFIIA

43
Q

Which eukaryotic protein factor of RNA polymerase III contains the TATA binding protein (TBP)?

A

TFIIB

44
Q

How many transcripts can be made by RNA polymerase III and TFIIA, TFIIB and TFIIC before the complex dissociates?

A

Multiple RNA transcripts can be made.

45
Q

How can the number of 5S rRNA that is transcribed by limited?

A

5S rRNA can bind to TFIIA to limit the amount of 5S rRNA that is transcribed. When 5S rRNA binds to TFIIA, TFIIA can’t bind to DNA and begin the RNA pol III elongation complex

46
Q

RNA polymerase III and its protein factors in eukaryotes bind to DNA in what order?

A
  • TFIIA
  • TFIIC
  • TFIIB
  • RNA polymerase III
47
Q

What do snRNA (small nuclear RNA) transcripts do in eukaryotes?

A

They are involved in intron splicing of mRNA

48
Q

Where is the TATA box found on DNA?

A

20-30 nt upstream of the start site for trnascription

49
Q

Eukaryotic RNA polymerase II recognizes promoters which have what 2 protein factors bound to it as part of a complex (what is the complex called)?

A

TFIID complex

  • TATA binding protein (TBP)
  • TBP-association factors (TAFs)
50
Q

True or false? All RNA polymerase II promoters have a TATA box recognized by TAFs?

A

False

Some but not all RNA polymerase II promoters have a TATA box, and it is recognized by TBPs

TAFs are TBP-associated factors that vary in number and composition depending on the type of gene and cell type, they may be involved in binding non-TATA promoter elements as well

51
Q

What sequence pattern is most often seen with the Pribnow box of prokaryotic transcription? Where on DNA is it seen?

A

The sequence pattern is something like TATAAT, it is NOT the TATA box though!

The Pribnow sequence is seen around -10 and the TATA box is seen around -35 region

52
Q

What is a DNA sequence that binds a transcription factor for RNA polymerase II transcription initiation in eukaryotes called?

A

A control element

53
Q

What is a control element of RNA polymerase II transcription initiation in eukaryotes that binds trans-acting factors and influence transcription of a particular gene but are far removed from that gene’s promoter called?

A

An Enhancer

54
Q

What are some general promoter and enhancer elements of of RNA polymerase II transcription in eukaryotes? (4)

A
  • TATA box (most common core promoter element)
  • CAAT box (common upstream element to TATA)
  • GC box (often found in TATA less promoters)
  • Octamer (Oct1 and Oct2 contain homeo domains)
55
Q

What are three special promoter and enhancer elements of RNA polymerase II transcription in eukaryotes?

A
  • HSE (heat shock response)
  • GRE (protein binds glucocorticoid elements)
  • TRE (protein binds thyroid hormones)
56
Q

What are the steps of RNA polymerase II transcription initiation in eukaryotes?

A
  • TBP (TDIID TATA binding complex) binds to TATA box with polymerase
  • The pre-initiation complex (PIC) also known as the closed complex is formed
  • DNA is unwound in the complex to make the open complex (Formation of the transcription bubble)
57
Q

What are cis-acting control elements of eukaryotic RNA polymerase II transcription?

A

Control elements that can be proximal to the promoter (eg. TATA box) or distal (eg. enhancer).

These can be general or specific to certain genes and gene families/

58
Q

What are trans-acting factors of eukaryotic RNA polymerase II transcription?

A

Transcription factors that can be general (such as TBPs) or specific (eg. some of the TAFs)

59
Q

What does the CTD domain of the largest RNA polymerase II subunit in eukaryotic transcription do?

A

When hydroxyl containing residues of CTD are phosphorylated, transition from initiation to elongation can occur

60
Q

What does the CTD domain of the largest RNA polymerase II subunit in eukaryotic transcription need? (what are essential to CTD domain structure?)

A

PTSPSYS heptad repeats

61
Q

List the five steps of mRNA synthesis by RNA polymerase II in eukaryotes

A
  1. RNA pol II recruited to DNA by transcription factors (preinitiation complex)
  2. Formation of transcription bubble (initiation complex)
  3. Phosphorylation of CTD during initiation to create elongation complex
  4. Elongation until the termination complex is reached
  5. Dephosphorylation of CTD and termination of transcription
62
Q

What is promoter clearance during mRNA synthesis by RNA pol III in eukaryotes?

A

When the elongation complex of RNA pol III and its phosphorylated CTD domain move downstream from TFIID complex on the promoter

63
Q

How does eukaryotic transcription terminate for RNA polymerase I?

A

Termination depends on a polymerase-specific termination factor that recognizes and binds specific DNA sequences at the end of the rRNA transcription unit

64
Q

How does eukaryotic transcription terminate for RNA polymerase II?

A

RNA polymerase III terminates after polymerizing a series of U residues. There is no obvious requirement for an upstream stem-loop structure

65
Q

How does eukaryotic transcription terminate for RNA polymerase III?

A

RNA polymerase II can terminate at multiple sites well beyond the 3’ end of the coding region.

An AUAAA sequence acts as a signal for endonuclease cleavage

A poly(A) tail is added by poly (A) polymerase and the CTD domain of RNA polymerase II makes contact with various mRNA processing factors

66
Q

What is the highly conserved upstream sequence that acts as a signal for endonuclease cleavage of mRNA transcript in eukaryotic transcription?

A

AAUAAA

67
Q

is mRNA cleaved 10 to 50 nucleotides downstream or upstream from the AAUAAA sequence?

A

downstream

68
Q

Describe the steps of termination for eukaryotic RNA polymerase II transcription

A

Pol II synthesizes RNA beyond the segment of the transcript containing the cleavage signal sequences, invluding the highly conserved upstream sequence AAUAAA

  1. Cleavage signal sequence is bound by an enzyme complex that includes an endonuclease, a plyadenylate polymerase and several other multi-subunit proteins involved in sequence recognition, stimulation of cleavage and regulation of the length of the poly(A) tail
  2. The RNA is cleaved by the endonuclease at a point 10 to 30 nucleotides downstream of the AAUAAA
  3. The polyadenlyate polymerase synthesizes the poly(A) tail to 80 to 250 nucleotides long, beginning at the cleavage site
69
Q

Which step of RNA pol II transcription termination requires ATP?

A

Polyadenylate polymerase synthesis of the poly(A) tail on mRNA at the cleavage site

70
Q

Is the poly(A) tail essential for mRNA’s translation into protein?

A

NO, this is an error that the textbook made.

71
Q

True or false? Most eukaryotic genes are regulated by single transcription-control elements. These are relatively simple in multicellular organisms.

A

False.

  • Regulated by multiple transcription-control elements
  • These are VERY complex in multicellular oganisms
72
Q

What is an UAS in eukaryotic yeast? What is it analogous to with mammals?

A

Upstream activity sequence

Analogous to enhancers in mammals

73
Q

What does the TATA box do?

A

Contributes to the level of transcription but also positions RNA polymerase II to initiation transcription

74
Q

What are three eukaryotic control elements?

A
  • Promoter-proximal elements (always)
  • Enhancers (always)
  • TATA box (often)
75
Q

What do co-activators (mediator complexes) do?

A

Proteins that bind/interact with both proximal and distal control-elements of eukaryotic gene’s promoter region to activate or repress transcription

These act as bridges between transcriptional activators and coactivator and the CTD of RNA polymerase II

76
Q

What are high mobility group proteins?

A

Non-histone chromatin-associated proteins that play a role in chromatin remodelling. These can bind directly to nucleosomes and DNA, promote DNA bending/looping

77
Q

What type of protein promotes DNA bending and looping?

A

High mobility proteins

78
Q

How do repressors repress transcription in eukaryotes?

A

These are proteins that

- Disrupt and prevent contacts between RNA pol II and activators/co activators (eg. mediator complex)

79
Q

What is linker scanning used for?

A

Used to pinpoint sequences with regulatory functions within a transcription control region

80
Q

What does linker scanning use to identify transcriptional control elements?

A
  • A set of constructs with contiguous overlapping mutations, cloned into a vector
  • A ‘reporter gene’ that codes for a protein that can be easily assayed (eg. a fluorescent protein)
81
Q

How does linker scanning work?

A

Different linker scanning (LS) mutants are used to scramble certain regions of DNA, one mutatoin per a molecule of DNA that is inserted into a vector (inserted into a host). The region scrambled is done serially down the sequence of DNA, so that activity of reporter-gene product is assayed and control elements can be identified at exactly the right spots. (see slides for example linker scanning essay results)

82
Q

How can non-denaturing polyacrylamide gel electrophoresis be used to identify DNA-protein interactions?

A

There is an decrease in electrophoretic mobility when DNA is bound by proteins

83
Q

What does a chromatin immunoprecipitation (ChIP) assay identify?

A

Identifies genomic DNA sites bound by proteins in vivo

84
Q

What are the steps of a chromatin immunoprecipitation (ChIP) assay?

A
  1. Cells are treated with formaldehyde to cross link DNA and associated proteins
  2. Cells are lysed and chromatin is isolated
  3. Chromatin is then sonicated or digested to 500-1000 bp fragments)
  4. Immunoprecipitate with antibody specific for protein of interest
  5. Reverse cross linking and purify DNA

Then…

  • The DNA can be used as a template for PCR, where a promoter region will amplify if protein of interest was bound there OR
  • Label and hybridize to microarray (ChIP on chip), if array represents entire genome, all sites in genome bound by the protein can be identified in one experiment

Nucleic Acid Biochem II (8 decks)

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