Lecture 11: Bacterial Transcription [G] but watch vids

Question 1

What is the central dogma?

Answer 1

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

Question 2

What is transcription?

Answer 2

The process of converting DNA to RNA.

Question 3

The top strand runs from 5’ to 3’ (left to right).

Answer 3

The bottom strand runs from 3’ to 5’ (right to left).

Question 4

At which levels can gene expression be regulated?

Answer 4

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

Question 5

Describe the sense strand

Answer 5

Matches the mRNA sequence (with U replacing T).

Question 6

Descibe the antisense strand

Answer 6

Template for mRNA synthesis; complementary to sense strand.

Question 9

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

Answer 9

Uracil

Question 10

What does what we call the sense strand depend on?

Answer 10

The context and the direction in which the gene is expressed

Question 11

How can cytosine produce uracil?

Answer 11

Cytosine can undergo spontaneous deamination to produce uracil.

Question 12

What happens when cytosine undergoes spontaneous deamination to produce uracil?

Answer 12

Mutations are potentially introduced

Question 13

Uracil isn’t allowed in DNA, but why is it allowed in RNA?

Answer 13

Uracil is allowed in RNA due to its transient nature, making RNA synthesis less error-sensitive.

Question 14

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

Answer 14

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 15

What are the 3 main types of bacterial RNA?

Answer 15

mRNA

rRNA

tRNA

Question 16

What does mRNA do in bacteria?

Answer 16

It encodes proteins

Question 17

What does rRNA do in bacteria?

Answer 17

Forms structural and functional components of ribosomes.

Question 18

What does tRNA do in bacteria?

Answer 18

Decodes mRNA by matching codons to amino acids during translation.

Question 19

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

Answer 19

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

Question 20

What does the operator control?

Answer 20

Whether the 5’ promoter is seen/accessed or not. If the promoter can be accessed, then transcription can be initiated.

Question 21

What does the 5’ promoter do ?

Answer 21

It attracts and binds RNA polymerase and other transcription factors, essentially initiating transcription.

Question 22

What does the 3’ terminator do?

Answer 22

The 3’ terminator is a DNA sequence located at the end of a gene or operon that signals the end of transcription. It ensures that RNA polymerase stops synthesizing RNA at the appropriate location and releases the completed RNA transcript.

Question 23

Why can transcription and translation occur simultaneously in bacteria?

Answer 23

Because bacteria have no nucleus

Question 24

What can the bacterial RNA polymerase be described as?

Answer 24

• 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 25

Describe the sigma subunit of RNA polymerase

Answer 25

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

Question 26

Where does the RNA polymerase bind to DNA?

Answer 26

At promoter sequences

Question 27

Is it true that the core RNA polymerase (without the sigma subunit) binds to DNA non-specifically and can slide?

Answer 27

Yes

Question 28

Why can the core RNA polymerase (without the sigma subunit) bind to DNA non-specifically and slide?

Answer 28

This behavior is an essential step in transcription because it allows the RNA polymerase to locate promoter regions efficiently.

Question 29

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

Answer 29

The RNA polymerase holoenzyme is directed to a gene promoter to initiate transcription.

Question 30

What is recognised as a promoter?

Answer 30

The specific sequence of bases

Question 31

Describe the DNA footprinting technique that was used to identify promoters.

Answer 31

• 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 32

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

Answer 32

Yes

Question 33

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

Answer 33

• 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
• Used to pinpoint promoter regions (-35 and -10), leaves a footprint

Question 34

The closer the promoter is to the concensus sequence…

Answer 34

… the stronger that promoter is

Question 35

What are the 2 promoter sequences?

Answer 35

TTGACA (-35)
TATAAT (-10)

Question 36

WHat is +1 usually?

Answer 36

An A or a G

Question 37

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

Answer 37

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 38

What does the asymmetry of the promoter sequence provide?

Answer 38

Directionality, ensuring that the DNA polymerase binds in the correct orientation.

Question 39

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

Answer 39

The sense strand

Question 40

In which direction is RNA built?

Answer 40

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

Question 41

What are the 3 stages of transcription in bacteria?

Answer 41

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 RNA polymerase continues synthesizing RNA 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 42

Describe initiation

Answer 42

• 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 success 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 43

Describe elongation

Answer 43

-The σ subunit disengages from the holoenzyme, and the core RNA polymerase continues synthesizing RNA by adding nucleotides in the 5’ to 3’ direction.

• The RNA polymerase unwinds the DNA ahead of it and rewinds it behind as it progresses

[ the core enzyme itself is RNA polymerase!!!!!]

Question 44

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

Answer 44

No

Question 45

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

Answer 45

Yes

Question 46

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

Answer 46

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 47

What are the 2 mechanisms of terminateing bacterial trasncription?

Answer 47

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 48

Describe Rho (ρ)-independent

Answer 48

• 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 49

Describe ρ -dependent

Answer 49

requires ρ/rho protein to bind to a specific site on RNA and use ATP to break the RNA:DNA duplex in the transcription bubble.

Question 50

What is Rifampicin?

Answer 50

an inhibitor of prokaryotic transcription

Question 51

How does Rifampicin inhibit prokaryotic transcription?

Answer 51

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 52

Why does RNA polymerase bend the DNA duplex?

Answer 52

Because bending allows the DNA duplex to be opened more easily. (strands to be split apart)

Question 53

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

Answer 53

No

Question 54

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

Answer 54

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

Question 55

Sigma factor binding is not static; it dynamically attaches and detaches to enable progression.

Answer 55

Sigma factor binding is not static; it dynamically attaches and detaches to enable progression.

Question 56

Processes like Rho helicase activity consume ATP, emphasizing the energy-intensive nature of transcription regulation.

Answer 56

Processes like Rho helicase activity consume ATP, emphasizing the energy-intensive nature of transcription regulation.