Lecture 11: Bacterial Transcription

Question 1

What is the central dogma?

Answer 1

The idea that we go from the DNA to RNA to proteins

Question 2

At which levels can gene expression be regulated?

Answer 2

At the level of transcription, at the level of translation, or both

Question 3

Why is the mRNA called the sense strand?

Answer 3

Because it’s produced from the sense strand of DNA

Question 4

What is the non-template strand called?

Answer 4

The sense strand

Question 5

What is the template strand called?

Answer 5

The ‘antisense’ strand

Question 6

In an RNA strand, what is the Thymine replaced with?

Answer 6

Uracil

Question 7

What does what we call the sense strand depend on?

Answer 7

The context and the direction in which the gene is expressed

Question 8

How can cytosine produce uracil?

Answer 8

Cytosine can undergo spontaneous deamination to produce uracil.

Question 9

What happens when cytosine undergoes spontaneous deamination to produce uracil?

Answer 9

Mutations are introduced

Question 10

How is the issue of cytosine undergoing sponatneous deamination to produce uracil corrected?

Answer 10

In DNA any Uracil will be removed by the enzyme uracil-DNA glycosylase, generating an abasic site, which is removed and repaired by DNA polymerase

Question 11

What are the 3 main types of bacterial RNA?

Answer 11

mRNA

rRNA

tRNA

Question 12

What does mRNA do in bacteria?

Answer 12

It encodes proteins

Question 13

What are the 3 types of bacterial RNA syntheisised by in E coli?

Answer 13

single RNA Polymerase (In eukaryotes, there is a separate RNA polymerase for each class).

Question 14

What does the operator control?

Answer 14

Whether the 5’ promoter is seen or not

Question 15

What does the 5’ promoter do ?

Answer 15

It attracts and binds RNA polymerase

Question 16

What does the 3’ terminator do?

Answer 16

it signals the stop point for transcription

Question 17

Why can transcription and nucleus occur simultaneously in bacteria?

Answer 17

Because bacteria have no nucleus

Question 18

What can the bacterial RNA polymerase be described as?

Answer 18

• A multi sub unit protein complex, containing an α - alpha subunit, a β - beta subunit. an ω - omega subunit, and a σ - sigma subunit.

Question 19

Describe the sigma subunit of RNA polymerase

Answer 19

It provides specificity and converts the core enzyme into a holoenzyme. It recognises specific promoters and initiates transcription.

Question 20

Where does the RNA polymerase bind to DNA?

Answer 20

At promoter sequences

Question 21

Is it true that the core RNA polymerase binds to DNA non-specifically and can slide?

Answer 21

Yes

Question 22

What happens when the sigma subunit binds to the core RNA polymerase?

Answer 22

The RNA polymerase holoenzyme is directed to a gene promoter.

Question 23

What is recognised as a promoter?

Answer 23

The specific sequence of bases

Answer 24

• Purify DNA
• Add holoenzyme

-Allow it to bind to a promoter

• Add DNase
• DNase will then digest all the accessible DNA
• The DNA inside the holoenzyme is inacessbible to the DNase.
• This protection provides us with a footprint that can help us identify promoters

Question 25

Is the aim of DNA footprinting partial degradation to generate a ladder of nicked intermediates?

Answer 25

Yes

Question 26

How do use DNA footprinting to work out what a promoter is and what it looks like?

Answer 26

• Label one end of DNA with a radioactive phosphate (32P)
• Add a very small amount of DNAse
• The DNAse should create nicks and this will cause partial degradation and a ladder of nicked intermediates will be generated.

-You can then undergo electropheresis, which will cause large fragments to go to the top of the gel and small fragments at the bottom of the gel.

• Add the holoenzyme RNA polymerase
• The RNA polymerase will bind to the promoter
• Add small amount of DNAse
• The RNA polymerase will mean that the DNA is inacessible to the DNase
• Where there’s no bands will tell you where the polymerase binds to the DNA
• This is representative of the places where the polymerase binds to the promoter

Question 27

The stronger the promoter is to the concensus sequence…

Answer 27

… the stronger that promoter is

Question 28

What are the 2 promoter sequences?

Answer 28

TTGACA (-35)
TATAAT (-10)

Question 29

WHat is +1 usually?

Answer 29

An A or a G

Question 30

Which 2 regions are protected from DNAse by RNA polymerase (promoter binding sites)?

Answer 30

One is centred around -10 bp (-10 sequence) from the start of transcription and the other centred around -35 bp (-35 sequence) from the start of transcription (+1).

Question 31

What does the asymmetry of the promoter sequence provide?

Answer 31

Directionality

Question 32

Which strand of DNA can the -10, -35 and +1 consensus sequences be found on?

Answer 32

The sense strand

Question 33

In which direction is RNA built?

Answer 33

he 5’ → 3’ direction: new nucleotides are added at the 3’ end, using the ANTISENSE strand as a template

Question 34

What are the 3 stages of transcription in bacteria?

Answer 34

Initiation, where RNA polymerase holoenzyme binds to the promoter (with the help of the sigma factor), opens the DNA double helix and starts to transcribe.

Elongation, where the σ subunit disengages from the holoenzyme, and the core enzyme continues to make new RNA, with the help of RNA polymerase, by adding nucleotides in the 5’ to 3’ direction.

Termination, where the RNA polymerase core enzyme dissociates from the DNA, and transcription halts. This will either be through the intrinsic or rho-dependant process.

Question 35

Describe initiation

Answer 35

• The core RNA polymerase binds to DNA non-specifically and can slide.
• A σ subunit binds to the core polymerase and directs the polymerase holoenzyme to a promoter. The RNA polymerase will then bind to the -10 and -35 regions of the promoter.
• The polymerase tries to unwind the DNA strands by pulling downstream DNA towards itself.
• This is called the scrunching the DNA and the DNA actually ends up becoming tighter and harder to unwind. (abortive initiation)
• Eventually there is sucess and the -10 region is opened, converting the CLOSED promoter complex to an OPEN promoter complex. Unlike the action of DNA helicase, this step does not involve the energy of ATP hydrolysis.
• 12 to 15 bp are unwound, from within the -10 region to position +2 or +3. The transcriptional start site is now exposed.
• RNA polymerase now makes an RNA copy from the template strand, using base pairing rules (G with C, A with U). Unlike DNA Pol, RNA polymerase does not require a primer.
• After about 10 nucleotides of RNA synthesis, the σ factor is exposed and disengages. The core RNA polymerase can now elongate the new RNA.

Question 36

Describe elongation

Answer 36

• During elongation, RNA polymerase is highly processive and adds nucleotides in the 5’ to 3’ prime direction
• The RNA polymerase unwinds the DNA ahead of it and rewinds it behind as it progresses

Question 37

Is the proofreading of RNA polymerase as good as in DNA polymerase?

Answer 37

No

Question 38

Do we tolerate a high level of errors with RNA polymerase?

Answer 38

Yes

Question 39

Why is such a high error rate tolerated with RNA polymerase?

Answer 39

DNA errors are transmitted to progeny cells: they are stringently repaired.
RNA errors mean that some transcripts may be mutated, but the majority are not.

If the transcript encodes a protein, them most of that protein will be fine, but a small subpopulation may be mutant – and can probably be tolerated.

Question 40

What are the 2 mechanisms of terminateing bacterial trasncription?

Answer 40

Rho (ρ)-independent

ρ -dependent

(In both cases, the functioning signals are recognised not in the DNA template, but in the newly synthesised RNA.
)

Question 41

Describe Rho (ρ)-independent

Answer 41

• A terminator sequence in the RNA is recognised. This sequence will end with a series of Uracils.
• This causes the RNA polymerase to pause, destabilizing its interaction with the DNA, leading to the release of the newly synthesized RNA and the dissociation of the RNA polymerase from the DNA.

Question 42

Describe ρ -dependent

Answer 42

requires ρ protein to break the RNA:DNA duplex in the transcription bubble

Question 43

What is Rifampicin?

Answer 43

an inhibitor of prokaryotic transcription

Question 44

How does Rifampicin inhibit prokaryotic transcription?

Answer 44

Rifampicin inhibits RNA Pol by binding tightly to the RNA exit channel.

It therefore affects initiation (but does not affect RNA Pol that is already at the elongation stage).

Question 45

Why does RNA polymerase bend the DNA duplex?

Answer 45

Because bending allows the DNA duplex to be opened more easily.

Question 46

After bacterial transcription, does further processing, like splicing, need to occur?

Answer 46

No

Question 47

Are the -35 and -10 promoter regions located upstream of the transcription site?

Answer 47

Yes, they are located before the transcription site and are recognised by RNA polymerase.