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BIOL 200 - Molecular Biology > Lecture 17 - Overview of Transcription and mRNA Synthesis > Flashcards

Lecture 17 - Overview of Transcription and mRNA Synthesis Flashcards

1
Q

Why is it important to evalute the levels of mRNAs in sample?

A

It is important because they are protein coding genes which play crucial roles in aspects of physiology, development, and medicine.

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2
Q

What process would one use to separate RNAs before transfering to a solid phase membrane?

A

Agarose Gel Electrophoresis

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3
Q

What alternative to radiolabeled probes can be used to evaluate polymorphism?

A

PCR

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4
Q

How can one purify mRNA from a complex sample of total RNA?

A

One can purify mRNA from a complex sample of total RNA by using affinity chromatography with a poly T column (poly dTTPs bound to agarose beads) which will bind by affinity to a poly (A) tail, which is only found on mature mRNA transcripts. One can then get rid of all the other RNAs and enrich a population of mRNAs by means of its poly (A) tail.

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5
Q

What method of mRNA purification is used in quantitative RT-qPCR?

A

Affinity Chromatography (with a poly T column)

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6
Q

What primer is used for reverse transcription in quantitative RT-qPCR? Why?

A

Poly T Primer

A poly T primer is used so that it can bind with the poly (A) tail on the mRNA.

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7
Q

If you perform quantitative RT-qPCR on a sample of 1000 mRNAs, how many cDNAs will be produced? Why?

A

1000 cDNAs will be produced.

This is because the poly T primer can bind to the poly (A) tail on any and all mRNAs present in the sample.

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8
Q

What is the end result of the RT process in RT-qPCR?

A

A collection of single stranded DNAs that are complementary to all the mRNAs of the given sample.

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9
Q

Describe what you do after you’ve obtained a library of cDNAs in RT-qPCR?

A
  • You prime a second round synthesis of the cDNA to create double stranded DNA that corresponds to the target mRNA in your sample.
  • You carry out a PCR reaction.
  • Using mathematics and algorithms, you quantify the amount of double stranded DNA amplified through PCR to get a quantitative value of how much cDNA you began with, which is equal to the amount of mRNA in the reaction.
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10
Q

When does RT-qPCR work best?

A

RT-qPCR works best if you have a known target mRNA and have no idea what is happening to the other RNAs in the sample.

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11
Q

What technique allows you to look at the whole transcriptome and observe how it adapts in physiological conditions?

A

RNA-seq

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12
Q

What are the four steps of RNA-seq?

A
  1. Form cDNA libraries corresponding to all mRNAs in the sample by performing single strand synthesis with RT to get a complementary DNA strand and then performing second strand synthesis using a DNA polymerase.
  2. Ligate the same adaptors for next generation sequencing (NGS) on each end of every double-stranded cDNA molecule.
  3. Amplify with PCR.
  4. Perform NGS on the cDNA library.
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13
Q

What organism does the DNA polymerase used in RNA-seq come from?

A

E. coli

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14
Q

What do the double-stranded cDNAs produced in the first step of RNA-seq represent?

A

They represent both the complexity and abundancy of the mRNAs present in the initial sample.

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15
Q

What is done with the raw data obtained from NGS in PCR-seq?

A

Computer alogirthms will recognize the sequences and then find corresponding genes in the genome database for the organism you’re studying. It will map all of those reads to individual regions of the genome, giving you a quantitative readout which indicates the amount of times it saw an mRNA that corresponds to that region (the read of your mRNA).

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16
Q

What does RNA-seq allow you to evaluate?

A

RNA-seq allows you to evaluate the levels of gene expression, or the levels of mRNAs of the genes present in the genome in different conditions, such as after a treatment, during a mutation, or over time.

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17
Q

Which mRNA quantification method would you use to quantify individual genes/one single gene product at a time?

A

Northern Blots or RT-qPCR

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18
Q

Which mRNA quantification method would you use to quantify mRNA levels of the entire transcriptome?

A

RNA-seq

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19
Q

How many cells and how many foreign cells (bacteria) do we have?

A

We have 30 trillion cells and 300 trillion foreign cells.

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20
Q

What is the process by which one can take similar cells to make tissues and organs that work together known as?

A

Development

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21
Q

What process uses DNA to make RNA?

A

Transcription

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22
Q

What enzyme does transcription require the activity of?

A

RNA Polymerase

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23
Q

What does RNA transcriptase do?

A

RNA transcriptase interacts with double stranded DNA and denatures it locally. It then goes along the single stranded DNA (3’ to 5’) and adds ribonucleotides that will form a growing complementary strand of RNA in a 5’ to 3’ direction

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24
Q

Which end of the nascent RNA strand produced in transcription will exit the RNA polymerase first?

A

5’

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25
Q

In transcription, what is the DNA strand that is read 3’ to 5’ known as?

A

Template Strand

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26
Q

In transcription, what is the DNA strand not used by RNA polymerase known as?

A

Non-Template or Coding Strand

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27
Q

Which DNA strand is very similar to the nascent RNA strand produced in transcription?

A

Non-Template/Coding Strand

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28
Q

At what rate does RNA polymerase function?

A

1000 nucleotides/minute

29
Q

How long does it take RNA polymerase to produce a mRNA of the Titan gene?

A

18-20 Hours

30
Q

What is the region before the 5’ of the transcription start site known as?

A

Promoter

31
Q

What are the two types of promoters found in DNA?

A

Proximal Promoters and Distal Promoters

32
Q

In transcription, what is the name of the DNA sequences that correspond to the actual nucleotides that will be incorporated into the nascent RNAs?

A

Coding Sequences

33
Q

What is the transcription start site also known as?

A

+1

34
Q

In which direction of the start site on the template strand is upstream? In which direction of the start site on the template strand is downstream?

A

3’

5’

35
Q

Does RNA polymerase move downstream or upstream on the template strand?

A

Downstream

36
Q

Where are enhancers found?

A

Enhancers are found far upstream or far downstream.

37
Q

What do enhancers do?

A

Enhancers affect transcription efficiency (by increasing the probability of transcription).

38
Q

What do enhancers do when chromatin is wound up?

A

When chromatin is wound up, enhancers interact with proteins aroun the promoter which will specify the transcription of a gene.

39
Q

What are the three stages of RNA transcription?

A

Initiation, Elongation, and Termination

40
Q

What happens during the initiation stage of RNA polymerase?

A

Polymerase interacts with the proximal promoter region. It locally denatures the DNA and then catalyzes the first phosphodiester bond in the formation of the RNA molecule.

41
Q

What is the most regulated and considered to be most important stage of RNA transcription?

A

Initiation

42
Q

What happens during the elongation stage of RNA transcription?

A

RNA polymerase leaves the promoter region (start site). Conformation changes occur within the polymerase that result in its stability on the DNA template. The enzyme goes 3’ to 5’ down the template strand (denaturing the DNA duplex and making a “transcription bubble”) to express the gene.

43
Q

What can RNA polymerase be referred to as during elongation of RNA transcription?

A

It can be called an engaged polymerase or an actively transcribing enzyme.

44
Q

What happens during the termination stage of RNA transcription?

A

The polymerase lets go of (dissociates from) the DNA template when it encounters the particular termination sequence (a stop site) and it terminate.

45
Q

What does RNA polymerase need in order to bring it to the appropriate transcribing position?

A

A Specificity Factor

46
Q

What would happen if a specificity factor is not present?

A

RNA polymerase would react with every little nick on DNA strands.

47
Q

What are specificity factors in bacteria known as?

A

σ (Sigma) Factors

48
Q

How can DNA binding proteins regulate the rate of RNA synthesis?

A

They regulate it by enhancing or impeding the RNA polymerase’s ability to bind to the promoter.

49
Q

How does the lac repressor impede transcription of the lac operon in E. coli?

A

Lac repressors bind tightly to the lac-o sequences, which impedes the RNA polymerase/σ70 complex’s interaction with the promoter region. It represses transcription until an allosteric change occurs (in the presence of lactose), changing the protein’s conformation and making it release the DNA strand so that transcription can occur.

50
Q

How does catabolite activator protein (CAP) enhance the activity of RNA polymerase (when looking at the lac operon)?

A

CAP will bind to cAMP molecules on the region of DNA before the lac operon promoter and will enhance transcription (which leads to an increase of lac operon expression). The CAP sense glucose through cAMP and activates transcription when glucose is low (since cAMP levels will rise, binding and activating more CAP).

51
Q

Which protein is responsible for the effect of glucose levels on lac operon transcription?

A

Catabolite Activator Protein (CAP)

52
Q

What is polycistronic mRNA?

A

Polycistronic mRNA is mRNA that codes for more than a single polypeptide.

53
Q

How did bacteria come to have polycistronic transcripts?

A

Through evolution, they arranged all genes that are involved in specific processes to be in the same transciptional region of DNA.

54
Q

What does the transcription of operons give rise to?

A

Polycistronic mRNAs

55
Q

What is an operon?

A

An operon is a group of several adjacent structural genes arranged under a common promoter and regulated by a common operator.

56
Q

Which types of cells can operons be found in?

A

Prokaryotic Cells

57
Q

What kinda of mRNA is produced in eukaryotes?

A

Monocistronic mRNA

58
Q

How many RNA polymerases are foudn in eukaryotes?

A

3

59
Q

What does eukaryotice RNA polymerase 1 make?

A

It is responsible for the transcription of all ribosomal RNAs (pre-rRNAs).

60
Q

What does eukaryotice RNA polymerase 2 make? What are these products important for?

A

It is responsible for all the protein coding genes (mRNAs), nuclear RNAs involved in splicing (snRNAs), small interfering RNAs (siRNAs) that are important for post transcriptional silencing and chromatin mediated repression, and micro RNAs (miRNAs), which are involved in post transcriptional regulation (translation control).

61
Q

What does eukaryotice RNA polymerase 3 make? What are these products important for?

A

It is responsible for all transfer RNAs (tRNAs) crucial in protein synthesis, one of the ribosomal RNAs (5S rRNA), some small nuclear RNAs involved in splicing (mainly the snRNA U6), the 7S RNA which is important as a signal recognition particle to insert polypeptides into the ER, and other small stable RNAs.

62
Q

What was used to prove that there is a difference in structure and function in the eukaryotice RNA polymerases? How?

A

Mushroom Toxin α-Amanitin

The RNA polymerases all had different sensitivities to the toxin, indicating they had differences in structure and function.

63
Q

Which RNA polymerase has similar structure between eukaroytes and prokaryotes?

A

RNA Polymerase 2

64
Q

What is “the clamp”? How does it work? Why is it important?

A

The clamp is an extended arm that is very typical of eukaryotic polymerases.

When eukaryotic polymerases are engaged, the clamp closes over the DNA.

It is important for the stability or the “processivity” of the RNA pol 2 over the DNA during elongation, as it allows it to transcribe for hours without falling off. It prevents the polymerase from dissociating from the DNA.

65
Q

Why is the structure and subunit composition fo the RNA polymerases high conserved and maintained between eukaryotic and prokaryotic organisms?

A

The structure originates from a common ancesteral organism.

66
Q

What main structural feature does RNA pol 2 have that 1 and 3 don’t?

A

RNA pol 2 has a tail, on its large subunit, with a CTD (carboxy terminal domain) associated with it.

67
Q

What is a CTD?

A

A carboxy terminal domain is a repeated unit that occurs 52 times (hepta-peptide repeat) and is phosphorylated (addition of a phosphte group) during transcription in vivo. It is required for every organism (yeast died without it) and appears to be an intrinsically disordered region for which we can’t really define a structure.

68
Q

During which stage of RNA transcription is CTD phosphorylated? What was studied to figure this out?

A

Initiation

This was found by studying the polytene chromosomes from the salivary gland of Drosophila.

69
Q

Which specific chromosomes are often used to identify where genes are being transcribed. Why?

A

The polytene chromosomes from the salivary gland of Drosophila are often used to identify where genes are being transcribed.

These genes are polytene (contain multiple copies of DNA), making them easier to look at.

BIOL 200 - Molecular Biology (22 decks)

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