Transcription

Question 1

Facts about transcription

Answer 1

• the transcription bubble stays the same size throughout
• unidirectional
• only one of the strands is being copied
• the RNA strand is only base paired for a short region of about 9 base pairs

(similarities to replication)
Enzyme: RNA Polymerase
Synthesis: occurs 5’ to 3’
Chemistry: 3’OH attacks phosphate on incoming nucleoside triphosphate

(differences to replication)

• synthesized strand is made out of ribonucleotides rNTPs
• RNA polymerase does not require a primer to initiate synthesis
• RNA polymerase can unwind the duplex DNA on its own
• The RNA product does not remain base-paired to the template DNA
• only one strand is transcribed
• transcription is much less accurate than DNA replication
• during transcription specific genes are selectively transcribed through a highly regulated processes whereas during DNA replication the entire genome must be replicated exactly one time each cell cycle

Question 2

What are the 5 core subunits in RNA Polymerase?

Answer 2

B B’ a’ a’’ w

Question 3

What are the similarities between RNA polymerases in prokaryotes and eukaryotes?

Answer 3

The CRAB CLAW configuration with an Mg ion in the middle required for catalysis along with the 5 core subunits.

Question 4

What is the chemistry of RNA Synthesis?

Answer 4

RNA polymerase has 2 Mg2+ ions. One shields the negative charges on the beta and gamma phosphates and the other shields the negative charges on the alpha phosphate along with the 3’OH.

Question 5

Upstream means…

Answer 5

toward the 5’ of a given sequence

Question 6

Downstream means…

Answer 6

toward the 3’ of a given sequence

Question 7

Direction of Transcription

Answer 7

RNA is always made in the 5’ to 3’ direction

Question 8

DNA template strand in comparison to RNA

Answer 8

template strand/antisense strand

-complementary and antiparallel to RNA

Question 9

DNA coding strand in comparison to RNA

Answer 9

coding strand/ sense strand

-same sequence as RNA except it has thymine instead of uracil

Question 10

Either strand of the gene can be used as a template strand. T/F

Answer 10

True. This depends on where the promotor is. It can be on the template or coding strand. This is different for different genes but for each unique gene, it is the same.

Question 11

Initiation of Transcription

Answer 11

RNA polymerase finds the promotor sequence and binds to it in the closed complex form which means the polymerase is bound to a closed double stranded DNA helix- this is easily reversible. Then it undergoes isomerization and a transition occurs to the open complex of single stranded DNA which involves a structural change in the polymerase. This is very stable and transcription is guaranteed to happen- essentially irreversible. this transition does not require energy ATP in prokaryotes
The beginning of transcription is actually inefficient because a consistent ~10nt strand needs to be made in order for transition to continue.

Question 12

Elongation of Transcription

Answer 12

A stable and efficient complex is formed with RNA polymerase and the DNA and RNA is made.

Question 13

What tells RNA polymerase where to start and stop transcription?

Answer 13

promotor and terminator sequences

Question 14

______ is required for transcription initiation to bring RNA polymerase specifically to promotor sequences.

Answer 14

The sigma factor

Question 15

RNA Polymerase Holoenzyme

Answer 15

Bacterial RNA
This is the bacterial core of RNA Polymerase + the initiation factor sigma. RNA polymerase holoenzyme only initiates transcription at promoter sites in vivo due to the presence of sigma

sigma 70 is the predominant sigma factor in E. Coli

Question 16

Features of a Promoter in Bacteria

Answer 16

Consensus Sequence for sigma70

1. UP- element: not always present but when it is, it is better
2. -35
3. -10 (TATATT)

• both the -35 and -10 are double stranded and can be recognized from either template strands
• they are (-) meaning they are upstream of the transcription start site

Question 17

How far apart are the preferred sequences of -35 and -10 for the sigma factor promoter?

Answer 17

17 base pairs

Question 18

Promoter “Strength”

Answer 18

This refers to how many transcripts can be initiated at a given time for a given gene

1. Pol must be able to bind efficiently to the promoter
2. Promoter must support isomerization
3. Pol must be able to escape the promotor

Question 19

What is the difference between strong and weak promoters?

Answer 19

• strong promoters have sequences that closely match the consensus and are highly expressed– may also contain the UP-element
• weak promoters dont match the consensus as well and are expressed at lower levels

Question 20

Which two subunits recruit RNA polymerase to the promoter?

Answer 20

sigma and alpha

1. alphaCTD binds to the UP element
2. alpha NTD contains sigma4 and sigma 2 that bind to -35 and -10 respectively

Question 21

Recognition of specific promoter elements involves __________insertion into DNA ______ and _______.

Answer 21

Recognition of specific promoter elements involves alpha helix insertion into DNA major groove and H-bonding.

Question 22

Alternative sigma factors recognize different promoter sequences- provides a form of transcriptional regulation. T/F

Answer 22

True

Question 23

How is the DNA melted into a single stranded form (open complex) when RNA polymerase binds to it?

Answer 23

It is driven by interactions between sigma70 region 2 and the -10 element. Two bases in the -10 element (non-template strand) flip out and insert into pockets of the sigma2 region. This occurs here because there is ATAT base pairing which only consists of 2 H-bonds.

Question 24

How many channels does RNA Polymerase have?

Answer 24

1. DNA entry
2. DNA exit
3. product RNA exit
4. entry for substrate rNTPs

Question 25

Transition from Initiation to Elongation

Answer 25

Abortive Initiation | -short

Question 26

Elongating Complex

Answer 26

- unwinds DNA in the front - reanneals DNA in the back - synthesizes RNA - dissociates RNA from the template - 2 proofreading functions **Notice how elongating RNA Polymerase has many jobs, compared to DNA replication -During elongation, polymerase uses a step mechanism and translocates the distance of one nt after each new rNTP is added. the size of the transcription bubble remains constant. - 3 step cycle: 1. binding of rNTP 2. catalysis of new phosphodiester bond 3. translocation

Question 27

What are the two proofreading functions for RNA Polymerase?

Answer 27

*these occur at the same active site so there is no movement of the transcript 1. Pyrophosphorolytic Editing: reverses the process by re-entering the PPi that was just removed 2. Hydrolytic Editing: H20 is used to removal bases containing the mismatch

Question 28

Termination of Transcription in Prokaryotes

Answer 28

Terminators: sequences in the DNA at the end of genes that trigger the elongating polymerase to dissociate from the DNA and release the synthesized RNA strand -2 forms: Rho-dependent and Rho-independent

Question 29

Rho transcription Termination Factor

Answer 29

- Hexameric ring-shaped protein with 6 identical subunits - ATPase activity - A member of the family of ATP-dependent hexametric helicases

Question 30

Rho-dependent termination

Answer 30

rut sites: rho utilization site- RNA elements of approx 40 nuts; initial rho binding site (upstream of the actual terminator) *sequence specific binding

Question 31

Rho-independent termination

Answer 31

Intrinsic Terminators Consist of: 1. a short inverted repeat of 20 nuts followed by... 2. a stretch of 8 A:U bps (A- template dna and U in RNA transcript) *A:U is the weakest base pair there is!