Molcular Biology 5 : Prokaryotic DNA transcription

Question 1

What is transcription ?

Answer 1

Transfer of the genetic information from DNA to RNA.

Question 2

What are the 3 steps of transcription ?

Answer 2

Initiation, elongation, termination.

Question 3

How many RNA Pol’s do prokaryotes have ?

Answer 3

Only ONE !

Question 4

What are the main characteristics of prokaryotic RNA Pol ?

Answer 4

• RNA polymerase synthesises RNA in a 5’-3’ direction
• RNA polymerase does not need a primer.
• The RNA product does not remain base-paired to the template DNA strand

Question 5

Which is the coding strand ?
Which is the non-coding strand ?
From which strand is mRNA synthesized ?

Answer 5

Coding strand = sense
Non-coding strand = template strand = antisense
mRNA is synthesized from the template strand

Question 6

What can we also call the coding strand and template strand ?

Answer 6

Coding strand = plus strand (Crick)

Template strand = minus strand (Watson)

Question 7

Are the transcription units in prokaryotes monocistronique or polycistronique ?
What does this mean ?

Answer 7

The transcription units in prokaryotes are often polycistronic (e.g the trp operon, the lac operon etc.)
Polycistronic mRNA refers to a messenger RNA which encodes two or more proteins.

Question 8

What are the sub-units of the E Coli Pol holoenzyme ?

What are their respective roles ?

Answer 8

Core polymerase: alpha-2betabeta’omega (aBB’w)
B’ : core polymerase subunit (DNA binding)
B : core polymerase subunit (polymerase active site)
a : scaffolding role
w : unknown role
The sigma factor:
sigma : types, role in transcription initiation (promoter recognition), main type is sigma(70)

Question 9

What does the 3D structure of RNA Pol resemble ?

Answer 9

A crab claw :

• B and B’ pincers, B flap
• the DNA double-helix enters through sigma-4, contacts sigma-3.2, sigma-3.1, sigma-2 and sigma-1.1.

Question 10

How is initiation activated ?

Answer 10

The bacterial RNA Pol positions itself on the promoter through the sigma factor.

Question 11

What are the key regions of the bacterial promoter ?

Answer 11

The -35 region : TTGACA (consensus sequence)

The -10 region : TATAAT (TATA box)

Question 12

Which sub-units recruit RNA Pol to the promoter ?

Answer 12

σ and α subunits recruit RNA polymerase to the promoter:

• alpha-CTD (C Terminal Domain) bind the UP-element
• alpha-NTD is bound to sigma, itself bound to sigma-4 and sigma-2
• sigma-4 binds the -35 region
• sigma-2 binds the -10 region

Question 13

With which nucleotide does transcription start with ?

Answer 13

ALWAYS A.

Question 14

What are the three phases of initiation ?

Answer 14

Promoter unbound.
Promoter bound to RNA Pol, closed complex.
Promoter bound to RNA Pol, promoter “melting” (open complex).

Question 15

What happens immediately after initiation ?

Answer 15

The initial transcribing complex is formed.
Elongation then proceeds.
RNA Pol then terminates and release RNA.

Question 16

How is transcription terminated in prokaryotes ?

Answer 16

Termination of transcription in prokaryotes involves termination signals.

Question 17

What are terminators ?

What are the 2 terminating methods in prokaryotes ?

Answer 17

Terminators: sequences which trigger the elongating polymerase to dissociate from the DNA and release the nascent RNA
2 ways :
- Rho dependent
- Rho-independent

Question 18

What is the Rho factor ?

Answer 18

• A six ringed protein
• Binds the RNA at termination recognition sites called rut sites.
• Binds to single stranded RNA.
• Doesn’t bind RNA being translated.
• Rho pulls the RNA out of the polymerase, or induces polymerase conformational change such that the polymerase terminates.

Question 19

What are the three steps of rho dependent termination of transcription ?

Answer 19

• Rho pursues RNA Pol
• HAirpin forms : RNA Pol pauses; rho catches up
• Rho causes termination

Question 20

How does Rho independent termination occur ?

Answer 20

• Intrinsic terminators,
• Do not involve other factors
• Short inverted repeat
• Stretch of 8 A-T base pairs

Question 21

Summarize the 3 steps of transcription by RNA Pol.

Answer 21

• Initiation : Involves recognition and binding of RNA Pol to the promoter (closed complex), promoter melting (open complex) and formation of stable ternary complex (transcription bubble).
• Elongation : Pol advances 3’-5’ down template strand, adding rNTPs to growing RNA chain.
• Termination : Transcription is terminated by signals within the RNA sequence. Pol releases completed RNA and dissociates from DNA.

Question 22

How can transcription be inhibited ?

Answer 22

Via binding of repressors binding to the operator region.

Question 23

What is an operator ?

Answer 23

In genetics, an operator is a segment of DNA to which a transcription factor binds to regulate gene expression by repressing it. The protein that does this is called a repressor.

Question 24

Which genes are under the control of the lac operator ?

Answer 24

lacZ (beta galactosidase), lac Y (lactose permease) and lacA (beta-galactoside transacetylase)

Question 25

How is the lac operon regulated when there is :

• lactose and glucose ?
• only glucose ?
• only lactose ?

Answer 25

Lactose + glucose = basal level of transcription
Only glucose = repressor bound to lacO, no transcription
Only lactose : CAP (Catabolic Activating Protein) bound to CAP site (upstream of promoter), RNA Pol bounds, activated level of transcription