RNA Transcription

Question 1

How does the 2’ hydroxl group on DNA vs. RNA contribute to the differences observed in stability of DNA and RNA.

Answer 1

In RNA the 2’ OH group is free so it can cyclize and weaken RNA –> less stable than DNA

Question 2

What are some major differences between DNA replication and transcription of DNA into RNA?

Answer 2

• DNA Replication requires a primer, transcription does not
• In transcription, only one strand is the template
• RNA polymerase has no proofreading function
  
  • A mistake in RNA is not as consequential as in DNA
  • RNA Pol has some ability to identify mistakes in base pairs and can remove mismatches from most 3’ base only

Question 3

When looking at 2 strands of DNA, which one is the template strand (antisense) an which one is the coding strand (sense)?

Answer 3

Template = 3’ to 5’

Coding = 5’ to 3’ –> identical to sequence of mRNA but with T’s –> U’s

Question 4

What is Long nc RNA?

Answer 4

Long non-coding RNA –> chromatin structure

Question 5

As the transcription bubble passes through the DNA, what happens to the DNA on either side of the bubble?

Answer 5

Upstream of bubble = negatively supercoiled

Downstream of bubble = positively supercoiled

Question 6

What is the promoter?

Answer 6

A region of DNA where RNA polymerase recognizes and binds to the DNA

Question 7

What is the terminator?

Answer 7

A sequence of DNA that signals the end of transcription

Question 8

Prokaryotes

The promoter contains two very important sequences that are involved in RNA polymerase recognition and binding.

• Where are these sequences?
• What are these sequences?
• What is their significance?
• How do they interact with RNA Polymerase?

Answer 8

• -10 and -35 positions
• -10 = TATAAT and -35 = TTGACA
• These are consensus sequences = sequences that are found in many DNA sequences of prokaryotes. The closer the DNA sequence is to these sequences at these positions, the more active the promoter and the more efficiently RNA polymerase binds to the promoter and initiates transcription
• -35: site where RNA polymerase recognizes promoter, -10: site where RNA polymerase “sits down” on DNA and starts unwinding DNA

Question 9

Prokaryotes

RNA polymerase has 4 subunits. What are they and what are their functions?

Answer 9

• Alpha: assembles the enzyme, recognizes the promoter (-35 position) and binds activators
• Beta: Binds to the DNA, RNA, and free ribonucleotides
• Beta prime: binds to promoter and unwinds DNA
• Sigma: promoter specificity

Question 10

Repressor

Answer 10

Molecule that prevents RNA polymerase from binding to the promoter and initiating transcription

Question 11

Activator

Answer 11

Molecule that facilitates RNA polymerase binding and initiating transcription

Question 12

Describe Rho Independent Termination

Answer 12

This occurs in prokaryotes. It is the most common type of termination in prokaryotes. Near the end of the transcript, there is a self-complementary region that allows the transcript to fold on itself and form a hairpin. The stem of this hairpin is rich in G=C so it is quite stable, and the hairpin causes RNA pol to pause. Immediately following the hairpin is a sequence of poly-Us which cause instability in the mRNA. These two factors combined cause the RNA to dissociate from the RNA pol and terminate transcription.

Question 13

Describe rho dependent termination.

Answer 13

Rho protein binds to the transcript at a location called rut site (C rich, G poor). Rho has ATPase activity, so it hydrolyzes ATP to power movement along the transcript in the 5’ to 3’. RNA polymerase pauses when RNA is made that is C rich and G poor so Rho has time to catch up to RNA polymerase. When Rho catches up to RNA polymerase it interacts w/ template DNA and new strand of RNA such that it unwinds the RNA/DNA hybrid in the transcription bubble leading to release of the mRNA

Question 14

What is a cis-acting element?

Answer 14

Any sequence of DNA that is not converted into any other form but that functions exclusively as a DNA sequence in situ, affecting only the DNA to which it is physically linked

Ex: promoter, terminator, 5’ UTR, 3’ UTR

Question 15

What is a trans-acting factor?

Answer 15

Encoded by a regulator gene that encodes a protein involved in regualting the expression of other genes. This is a protein that binds to particular sites on DNA and can either have a positive or negative effect on the expression of that gene.

Ex: Transcription factors

Question 16

What is a significant difference between eukaryotic and prokaryotic gene structure?

Answer 16

Prokaryotes are polycistronic (one gene encodes mulitple products)

Eukaryotes are monocistronic (one gene encodes a single product)

Question 17

Eukaryotes have 3 different kinds of RNA polymerase. What are they?

Answer 17

RNA Polymerase 1 - synthesis of preribosomal RNA (pre-rRNA), which contains precursors for ribosomal rRNAs

RNA Polymerase 2 - synthesis of mRNA, miRNA, small RNAs

RNA Polymerase 3 - synthesis of tRNAs

Question 18

Eukaryotes

RNA polymerase 2 recognizes the _____ at position ____, which is followed by the ____ sequence at position ____. This is necessary to begin transcription.

Answer 18

TATA box

-30

INR

+1

Question 19

Eukaryotes

Can RNA pol II bind to the DNA by itself?

Answer 19

No - RNA Pol II has no affinity for the promoter by itself, it requires the help of transcription factors

Question 20

Eukaryotes

Describe the major steps in RNA Pol II initiation.

Answer 20

• TF - D binds to TATA
• TF - A stabilizes TF - D
• TF - B binds to TF - D creating place for RNA Pol II to bind
• TF - F helps RNA pol II associate w/ promoter
• TF - E and TF - H (Kinase and helicase activity) bind to Pol II and allow it to be released from promoter to continue transcription

Question 21

Eukaryotes

Not all organisms / genes have a TATA box. What happens with regards to initiation if there is no TATA box?

Answer 21

Same general TFs are needed, but they bind to the INR instead

Question 22

Eukaryotes

Housekeeping genes may not have a TATA or an INR sequence.

1. What does RNA Pol II do with respect to these genes?
2. What is the result with regards to rate of transcription?
3. What is a CpG island?

Answer 22

1. RNA Pol II can still initiate transcription, infact these genes are continuously expressed and are often important to metabolism so the cell wants to have them around at all times
2. The rate is much slower so there is a constant but low level of expression
3. A CpG island is an area of DNA that is rich in CG bonds upstream of the start site. Methylation occurs on Cytosine residues, but the CpG islands will never be methylated b/c they are upstream of housekeeping genes and methylating them would reduce their transcription which would likely have severely negative consequences for the cell.

Question 23

Eukaryotes

What are some means by which transcription factor activity can be regulated?

Answer 23

• Covalent modification (phosphorylation, acetylation, ubiquitination)
• Binding to ligands
• Dimerization

Question 24

Eukaryotes

How does transcription terminate?

Answer 24

• RNA Pol II extends ~ 1000 nucleotides beyond end of mature mRNA
• Pauses when a bound mediator protein recognizes termination sequence (AAUAAA)
• RNA is cleaved at the Poly A site, anything downstream of site is degraded
• RNA Pol II dissociates from DNA and RNA
• Poly A tail is added to 3’ end mRNA