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Bio 323 - Exam Two > Transcription and Transcriptional Regulation > Flashcards

Transcription and Transcriptional Regulation Flashcards

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1
Q

What are some uses of PCR?

A
  • detection of infectious organisms – such as microbes and viruses
  • forensics and disease diagnosis
  • detection of gene expression (RT-PCR)
  • protein engineering and DNA analysis
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2
Q

What determines the ends of the product of PCR?

A

The 5’ ends of the primer used

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3
Q

Northern blotting steps

A
  1. isolate total RNA
  2. denature and separate RNA
    by electrophoresis
  3. transfer to membrane
  4. Detect using labeled (radioactive)
    antisense probe to hybridize
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4
Q

TBP:

A

TATA-box Binding Protein, eukaryotes

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5
Q

What does TFIIH do?

A

acts as both a DNA helicase and a kinase

during initiation

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6
Q

Does an RNA strand have to be completely made before a new one is started?

A

No. The synthesis of additional RNA molecules can be started before previous RNA molecules are completed

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7
Q

Does RNA polymerase need a primer?

A

No.

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8
Q

What is the proofreading mechanism of RNA polymerase?

A

If an incorrect nucleotide is added to the growing RNA chain, the polymerase can back up, and the active site of the enzyme can perform an excision reaction that resembles the reverse of the polymerization reaction, except that a water molecule replaces the pyrophosphate and a nucleoside monophosphate is released.

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9
Q

snRNAs

A

small nuclear RNAs, function in a variety of nuclear processes, including the splicing of pre-mRNA

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10
Q

What is the sigma factor?

A

a subunit that associates with the RNA core enzyme and assists in beginning DNA transcription in prokaryotes. bind specific promoter elements

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11
Q

transcription bubble

A

the region of unpaired DNA which is stabilized by the binding of the sigma factor to unpaired bases on one of the exposed strands

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12
Q

How is transcription terminated in prokaryotes?

A

string of A-T nucleotide pairs which fold into a hairpin. The formation of the hairpin helps to disengage the RNA transcript from the active site.

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13
Q

Are all termination sequences the same?

A

no, there is variation, as long as they form hairpins its chill

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14
Q

If an RNA sequence is 5’-GUGUACACAU-3’, what is the sequence of its DNA template?

A

5’-ATGTGTACAC-3’

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15
Q

The strand in dsDNA used as a template for RNA synthesis depends on:

A

the orientation of the promoter

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16
Q

The majority of RNA transcripts in cells are _____ and predominately _____

A

The majority of RNA transcripts in cells are noncoding RNAs and predominately rRNA

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17
Q

Which of the eukaryotic RNA polymerases is responsible for mRNA synthesis?

A

RNA polymerase II

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18
Q

Which is more accurate, DNA or RNA polymerase?

A

DNA polymerase

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19
Q

Which is more processive, DNA or RNA polymerase?

A

RNA polymerase

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20
Q

Genes that are expressed in practically all cells in the human body include:

A

tRNAs

RNA polymerase

histones

ribosomal proteins

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21
Q

Restriction nucleases cut

A

DNA at specific sequences

22
Q

Main differences between RNA polymerase II and bacterial RNA polymerase

A

1) While bacterial RNA polymerase requires only a single transcription initiation factor (sigma) to begin transcription, eukaryotic RNA polymerases require many such factors, collectively called the general trasncription factors
2) Eukaryotic transcription initiation must take place on DNA that is packaged into nucleosomes and higher-order forms of chromatin structure, features that are absent from bacterial chromosomes

23
Q

How far is the TATA box from the transcription start site?

What binds to it?

A

About 25 nucleosides upstream from the transcription start site

TFIID

24
Q

What are transcriptional activators?

A

gene regulatory proteins that bind to specific DNA sequences to help attract RNA polymerase II to the start point of transcription

25
Q

Where do transcriptional activators bind?

A

enhancers

26
Q

What is a mediator?

A

a large protein complex which allows the activator proteins to communicate properly with the polymerase II and the general transcription factors.

27
Q

What is the last step of transcription initiation?

A

the recruitment of chromatin-modifying enzymes

28
Q

Steps at which eukaryotic gene expression can be controlled

A

1) transcriptional control
2) RNA processing control
3) RNA transport and localization control
4) translation control
5) mRNA degradation control
6) protein activity control

29
Q

What do restriction nucleases do?

A

facilitate isolation and manipulation of DNA at specific sites

30
Q

How does vector cloning work?

A

restriction endonuclease is added to cleave DNA and then the DNA fragment to be cloned is incorporated into the strand/

31
Q

DNA library

A

a collection of cloned plasmid molecules

32
Q

reverse transcriptase

A

makes DNA from mRNA

33
Q

Order of binding for prokaryotes

A

1) TBP binds to TATA box
2) TFIIB binds to complex
3) Pol II binds to complex
4) TFIIH binds to complex, acts as both a DNA helicase and a kinase during initiation
5) release of general factors except TBP and Pol II, C-terminus of Pol II becomes phosphorylated.

34
Q

what does a gene consist of?

A

promoter and transcription unit

35
Q

What do repressors bind?

A

silencers

36
Q

prokaryotic vs eukaryotic location of binding sites

A

Prokaryotic: typically close to the promoter

Eukaryotic: can be far or close, upstream, downstream, in the transcription unit itself

37
Q

What does it mean for something to be a cis-regulatory sequence?

A

“Cis”: the DNA element is on the same piece of DNA as the gene promoter it regulates

38
Q

How do proteins recognize specific DNA sequences?

A

By recognizing H-bond donors, acceptors, or methyl groups (specificity)

39
Q

Which contacts are likely important for the affinity of DNA binding proteins?

A

contacts between the binding protein and

1) DNA phosphates
2) DNA deoxyriboses
3) DNA bases

40
Q

What activates CAP in the lac operon?

A

cAMP

41
Q

How does lactose activate lac operon transcription?

A

1) lactose becomes allolactose

2) allolactose then activates lac operon transcription by activating the lac repressor

42
Q

How is glucose related to adenynyl cyclase?

A

Glucose inhibits the activity of adenylyl cyclase

43
Q

What does adenynyl cyclase do?

A

ATP —> cAMP

44
Q

When is the lac operon transcribed?

A

lac operon is only transcribed when lactose is present and glucose is not

45
Q

cis-regulatory elements vs promoters

A

cis-regulatory elements are specific sequences bound by transcriptional regulators,

promoters are common between genes and are where general transcription factors and RNA polymerase binds

46
Q

how do eukaryotic transcriptional regulators act?

A

They act indirectly to affect RNA polymerase-promoter association

47
Q

where would an enhancer bind?

A

cis-regulatory sequence

48
Q

What complexes formed on transcription regulators act as activators?

A

histone modifiers (acetyltransferases, kinases, methyltransferases) and histone remodelers

49
Q

What complexes formed on transcription regulators act as modifiers?

A

deacetylases, methyltransferases

50
Q

How are regulators regulated?

A

1) expression levels
2) activity levels
3) localization

51
Q

examples of reporter genes

A

epitope-tagged proteins (HA; Western)

luciferin (light emission when treated with luciferase)

GFP or GFP-tagged proteins (fluoresce green under UV light)

52
Q

what can gene expression constructs be used for?

A

Can be used to express:

1) a protein or RNA of interest (ex. HAtagged
Upf1)

2) a reporter gene controlled in expression by an element of interest
(gene product is easily measured to “report” on transcriptional activity)

Bio 323 - Exam Two (4 decks)

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