Molcular Biology 5 : Prokaryotic DNA transcription Flashcards
What is transcription ?
Transfer of the genetic information from DNA to RNA.
What are the 3 steps of transcription ?
Initiation, elongation, termination.
How many RNA Pol’s do prokaryotes have ?
Only ONE !
What are the main characteristics of prokaryotic RNA Pol ?
- 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
Which is the coding strand ?
Which is the non-coding strand ?
From which strand is mRNA synthesized ?
Coding strand = sense
Non-coding strand = template strand = antisense
mRNA is synthesized from the template strand
What can we also call the coding strand and template strand ?
Coding strand = plus strand (Crick)
Template strand = minus strand (Watson)
Are the transcription units in prokaryotes monocistronique or polycistronique ?
What does this mean ?
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.
What are the sub-units of the E Coli Pol holoenzyme ?
What are their respective roles ?
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)
What does the 3D structure of RNA Pol resemble ?
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.
How is initiation activated ?
The bacterial RNA Pol positions itself on the promoter through the sigma factor.
What are the key regions of the bacterial promoter ?
The -35 region : TTGACA (consensus sequence)
The -10 region : TATAAT (TATA box)
Which sub-units recruit RNA Pol to the promoter ?
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
With which nucleotide does transcription start with ?
ALWAYS A.
What are the three phases of initiation ?
Promoter unbound.
Promoter bound to RNA Pol, closed complex.
Promoter bound to RNA Pol, promoter “melting” (open complex).
What happens immediately after initiation ?
The initial transcribing complex is formed.
Elongation then proceeds.
RNA Pol then terminates and release RNA.
How is transcription terminated in prokaryotes ?
Termination of transcription in prokaryotes involves termination signals.
What are terminators ?
What are the 2 terminating methods in prokaryotes ?
Terminators: sequences which trigger the elongating polymerase to dissociate from the DNA and release the nascent RNA
2 ways :
- Rho dependent
- Rho-independent
What is the Rho factor ?
- 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.
What are the three steps of rho dependent termination of transcription ?
- Rho pursues RNA Pol
- HAirpin forms : RNA Pol pauses; rho catches up
- Rho causes termination
How does Rho independent termination occur ?
- Intrinsic terminators,
- Do not involve other factors
- Short inverted repeat
- Stretch of 8 A-T base pairs
Summarize the 3 steps of transcription by RNA Pol.
- 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.
How can transcription be inhibited ?
Via binding of repressors binding to the operator region.
What is an operator ?
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.
Which genes are under the control of the lac operator ?
lacZ (beta galactosidase), lac Y (lactose permease) and lacA (beta-galactoside transacetylase)
How is the lac operon regulated when there is :
- lactose and glucose ?
- only glucose ?
- only lactose ?
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
