Transcription

Question 1

What is transcription?

Answer 1

Using DNA as a template to synthesize RNA

Question 2

Why do we need to say that a gene is a sequence of nucleic acids that encodes a functional product, not a sequence of DNA that encodes a protein?

Answer 2

RNA viruses exist, and they have RNA that encodes a functional product. There are also genes that encode tRNA and rRNA (and other types of RNA), so the end product isn’t always a protein

Question 3

What is the primary enzyme used in transcription?

Answer 3

RNA polymerase

Question 4

What is used as a template for transcription?

Answer 4

DNA

Question 5

What are the subunits being assembled during transcription?

Answer 5

NTPs

Question 6

What are the 3 key steps in transcription?

Answer 6

Initiation, elongation, termination

Question 7

Which direction is the coding strand of DNA read during transcription? How does the mRNA look compared to this strand?

Answer 7

5’ to 3’. The mRNA produced is identical to the coding strand with all the thymines replaced with uracil

Question 8

Which direction is the template strand of DNA read during transcription? How does the mRNA look compared to this strand?

Answer 8

3’ to 5’. The mRNA produced is complementary to the template strand since this is the strand that RNA polymerase reads

Question 9

What defines the beginning of transcription?

Answer 9

The promotor, where RNA polymerase binds

Question 10

What is the +1 nucleotide?

Answer 10

Where transcription is initiated and the RNA transcript begins to be assembled

Question 11

What defines the end of transcription?

Answer 11

The terminator

Question 12

What defines the promotor regions in prokaryotes?

Answer 12

The two consensus sequences at -10 and -35 with high affinity for the sigma subunit of RNA polymerase

Question 13

Why is the sigma subunit important? What happens to RNA polymerase without it?

Answer 13

The sigma subunit is what physically binds to the two consensus sequences. Without it, RNA polymerase is able to elongate the strand of mRNA but loses the specificity of binding to the promotor and will be able to bind to anything

Question 14

What is RNA polymerase called when the sigma subunit is present?

Answer 14

The RNA polymerase holoenzyme

Question 15

What are the 4 steps of transcription initiation in prokaryotes?

Answer 15

1. The RNA polymerase holoenzyme binds to DNA
2. Binds tighter at the promotor
3. Tight binding promotes local melting of the DNA into single strands
4. Transcription begins and RNA polymerase continues to unwind the double helix as it continues to transcribe

Question 16

Does RNA polymerase need a primer?

Answer 16

No, it can synthesize de novo

Question 17

What is happening inside of RNA polymerase during elongation in prokaryotes? What happens to the DNA after it is transcribed? What is happening to the mRNA being synthesized?

Answer 17

Only a short stretch of synthesized mRNA remains base paired to to DNA inside RNA polymerase. The DNA reanneals as a double helix after it leaves RNA polymerase and the synthesized mRNA protrudes out of the enzyme

Question 18

What is processive transcription? What contributes to RNA polymerase being able to processively transcribe?

Answer 18

Processive transcription is saying that RNA polymerase is good at continuously catalyzing the reaction, so it won’t fall off until it is supposed to. Tight binding to DNA, RNA, and the DNA/RNA hybrid helix allows transcription to be processive

Question 19

How does RNA polymerase proofread? What will make it stop transcribing to proofread?

Answer 19

RNA polymerase will occasionally stop and backtrack about 5 base pairs to proofread. If it finds a mismatch, it uses endonucleolytic cleavage to cut off the strand and then resynthesizes it. If no mismatch is found, it goes back to where it was and keeps transcribing. A weak DNA/RNA hybrid helix will cause it to stop from a lack of hydrogen bonds

Question 20

What happens to the DNA/RNA hybrid helix during transcription termination?

Answer 20

It gets disrupted, destabilized and unwound

Question 21

What is Rho?

Answer 21

A helicase involved in transcription termination in prokaryotes

Question 22

What happens in Rho dependent termination?

Answer 22

1. Rho binds to the rut site in the RNA transcript
2. It follows RNA polymerase along the transcript
3. When RNA polymerase transcribes the terminator sequence, it will pause and Rho can catch up
4. Rho unwinds the RNA/DNA hybrid helix

Question 23

How does the terminator sequence cause RNA polymerase to pause in Rho dependent termination?

Answer 23

The terminator sequence forms a stemloop, which physically pulls on the RNA still in RNA polymerase and weakens the RNA/DNA hybrid helix. The weakened duplex from the stemloop causes the pause

Question 24

How does intrinsic termination work in prokaryotes?

Answer 24

A GC rich sequence gets transcribed which forms a stemloop, then a polyU sequence is transcribed. These two things destabilize the DNA/RNA hybrid helix enough for RNA polymerase to stop and fall off

Question 25

How is RNA polymerase different between eukaryotes and prokaryotes?

Answer 25

Eukaryotes have different versions of RNA polymerase that create different kinds of RNA, while prokaryotes only have the one. The core of RNA polymerase is conserved from prokaryotes to eukaryotes, but eukaryotes have no homolog for the sigma subunit

Question 26

Can eukaryotic RNA polymerase initiate transcription on its own?

Answer 26

No. It requires general and specific transcription factors to initiate transcription

Question 27

What is the difference between general and specific transcription factors?

Answer 27

General transcription factors are always required for transcription initiation at every gene in eukaryotes. Specific transcription factors regulate transcription at certain genes

Question 28

What do the general transcription factors do to initiate transcription in eukaryotes?

Answer 28

They bind to the promotor sequences and recruit RNA polymerase II. They also contain helicase activity that promote local melting

Question 29

What are the steps in transcription initiation in eukaryotes?

Answer 29

1. The general transcription factor recognize and bind to the promotor sequence 2. They recruit RNA polymerase 3. One of the general transcription factors phosphorylates the tail of RNA polymerase and recruits the elongation factors 4. DNA melting occurs and transcription begins

Question 30

Are the promotor sequences exactly the same in all eukaryotic promotors?

Answer 30

No, there are lots of possible sequences, and each promotor will have a different combination. Each sequence is bound to by one specific general transcription factor

Question 31

What is the TATA box? What binds to it?

Answer 31

A promotor sequence that is commonly found in eukaryotic promotors. TBP binds to it - TATA binding protein

Question 32

Are the general transcription factors involved in elongation in eukaryotes?

Answer 32

No, elongation factors are there instead. The general transcription factors are only for initiation and will stay bound at the promotor

Question 33

What do the elongation factors do?

Answer 33

RNA processing: splicing, capping, polyadenylation. One also does endonucleolytic cleavage after the terminator is transcribed

Question 34

What happens when the terminator sequence is transcribed in eukaryotes?

Answer 34

1. An elongation factor cuts the mRNA right after the terminator sequence 2. RNA polymerase continues to transcribe 3. Rat1 (an exonuclease) degrades the excess RNA until it reaches the RNA/DNA hybrid helix 4. The hybrid helix is disrupted and RNA polymerase falls off