Transcription Flashcards
What is gene expression?
The process of decoding DNA into protein
Why do we use E. coli as a model system for gene expression?
Bacteria are key human pathogens
The bacterial gene expression machinery is the target for some antibiotics
E. coli us an important host for the production of ‘recombinant’ proteins for research and industrial/medical purposes
Understanding the process of gene expression and its control in E. coli provides a framework for understanding it in more complex organisms.
What is transcription?
Transfer of genetic information from ds DNA to ss RNA (mRNA)
What is the template and non-template strand?
DNA acts as a template – the template strand is the bottom strand (3’ to 5’)
The top strand is the non-template strand (5’ to 3’)
What is the sense and the anti-sense strand?
The RNA has a strand that is complementary to the template strand and therefore the template strand has the opposite sense to the RNA; it is the antisense strand.
Because the top strand of the DNA has the same sequence as the RNA, it can be known as the sense strand, although it is NOT the template strand.
Sense = 5' to 3' Nonsense = 3' to 5'
What makes up transcription in E.coli?
Promotor
Transcribed region
Terminator
What does the promotor do in E.coli?
Promoter dictates when and to what level the gene is going to be transcribed, and where the polymerase will bind and start transcription.
It is the key region in transcription because it dictates how level of efficiency of the process.
What is the transcribed region in E.coli?
In prokaryotic cells, such as E. coli the resulting mRNA in the transcribed region can be called polycistronic, which means that the mRNA can be decoded into more than one protein.
This generally does not happen in eukaryotic organisms.
What does the terminator do in E.coli?
Stops the process of transcription
What is the E. coli sigma-70 promotor?
A sequence of about 60 base pairs immediately upstream of the transcription start site
Start site of transcription is E. Coli is always +1.
The promoter contains six base pair sequences, located about 10 base pairs upstream of the transcription site and 35 bp upstream, termed the -10 and -35 sequences.
What are the -35 and -10 sequences in the E.coli promotor sigma-70?
These are the consensus sequences for the promoter.
The closer a promoter is to these ideal, consensus sequences, the stronger, and more efficient that promoter will be.
The more changes it has, the weaker and less efficient it will be.
This is the primary level of the control of gene expression in E. coli.
What makes an efficient promotor?
An efficient promoter will give rise to lots of transcripts which will give rise to lots of proteins.
An inefficient promoter produces fewer transcripts and thus fewer proteins.
How are consensus sequences determined?
By sequence comparison
There is not a single E. coli promotor that has the exact consensus sequence
The closer you are the the perfect sequence, the stronger and more efficient the promoter, the more RNA and the more protein therefore produced.
Therefore, the sequence of the promoter will dictate the efficiency of the transcription
What is core RNA polymerase composed of?
2 alpha subunits
1 beta subunit,
1 related beta prime subunit and
1 omega subunit
What does core RNA polymerase do?
Core polymerase can catalyse the process of RNA synthesis of elongation; it can make RNA chains.
However, core polymerase cannot efficiently recognise and bind to a promoter
What allows core RNA polymerase to recognise and bind to a promoter?
It needs a sigma factor
The core polymerase plus the sigma factor gives you the form of the enzyme known as the holoenzyme which is the form of the enzymes that is able to recognise and efficiently bind to the promoter
Core + sigma factor = holoenzyme
What is a closed complex?
Initial binding of RNA polymerase to promoter
The recognition is completed by the sigma factor which binds directly to the -35 region and the -10 region.
After the formation of the closed complex, there is a transition to the formation of the open complex
What is the open complex?
The open complex is much more stable, and is characterised by the melting (un-base pairing) of a bubble of DNA (transcription bubble) over the transcription start site. The DNA is effectively opened up
During the process of open complex formation, the template strand gets hauled into the catalytic site of the enzyme, where the template strand is used to make ssRNA.
This results in the formation of the first phosphodiester bond between the first two nucleotides, and the cycle is repeated to form an mRNA strand in a 5’ to 3’ direction
How does chain formation occur?
5’ to 3’
Catalysed by active site in core polymerase and requires
• magnesium
• nucleophilic attack by hydroxyl group on alpha subunit
• release of pyrophosphate
What happens when there are mistakes in elongation?
RNA polymerase has no proofreading function like DNA polymerase does. But it can backtrack giving you a similar proofreading function
RNA polymerase is a lot less accurate and much slower than DNA polymerase.
If there is a mistake in the RNA it is not passed onto from generation to generation as it is the template strand that is going to be turned over whereas DNA is passed on to generation to generation so a mistake would be passed on meaning RNA does not have to be as accurate.
Therefore, RNA polymerase can afford to make a lot more mistakes than DNA polymerase.
What are the 2 types of termination?
Factor independent
Rho termination
What is factor independent termination?
Series of 4-10 consecutive A-T base pairs
A GC rich region with a palindromic sequence that immediately proceeds the series of A-T base pairs
Transcript will adopt the secondary structure where you H bonds pairing between the G C sequences to form the base paired stem with a non-base paired loop at the top.
After the Gs and Cs in the transcript, there is a string of Us which base pair very poorly with the DNA, so the interaction between the DNA and RNA has weakened
What does the formation of the hair pin bend in factor independent termination do?
The weakening of the interaction between the transcript and the template
The pausing of RNA polymerase.
As a consequence, this favours the dissociation of the transcript which causes the RNA polymerase to fall off.
What is Rho dependent termination?
Rho factor is composed of 6 identical subunits - RNA is in the middle in the hole
Rho is a helicase that unwinds RNA-DNA and RNA-RNA complexes
Powered by the hydrolysis of ATP
• Rho catches up because RNA polymerase pauses at the terminator site.
• Rho unwinds the RNA DNA complex leading to loss of the transcript so RNA can dissociate off the transcript and the polymerase is released
How do cells react to changes in the environment?
By changing:
• The sets of genes expressed
• The level of which they are expressed
What are the 2 strategies for regulating transcription initiation?
Repression
Activation
What happens in repression?
Repression can be thought of as a break
RNA polymerase bound to promoter is blocked by negatively charged repressor (removal of repressor allows transcription to occur)
It either prevents it from binding or from initiating transcription.
Loss or repression may be referred to as induction or depression
What is activation?
Often involves a weak promoter
Holoenzyme might not find it efficient to initiate transcription at this weak promoter, so there is none or very little transcription.
The positively active factor will bind to the polymerase and compensate for the weak promoter and the poor, non-consensus elements.
What is the lac operon?
Composed of a promoter (Plac)
The lac structural genes are lacZ lacY and lacA
Polycistronic mRNA.
E. coli can use lactose as a sole source of carbon but lactose is a disaccharide so there is effort with taking it and breaking it down; glucose is a more preferable carbon source as it is a monosaccharide.
What is the lac repressor?
Product of the lacI gene
360 amino acid protein
Forms a homotetramer
Binds to the lac operator
What is the lac operator?
The primary lac operator is found just downstream of the -10 region of the lac promoter, so it spans the transcription start site.
Has a very high affinity to the lac repressor - bind very tightly
In the absence of lactose, the lac repressor will always be occupying this site.
Doesn’t block the RNA polymerase from binding to the promoter, but prevents it initiating transcription.
How does the lac repressor bind to the lac operator?
The lac repressor has four DNA binding domains, two bind to the lac operator and the other two bind to secondary operator sites
Full repression requires one or both of O2 and O3 (secondary operator sites) to be occupied as well as O1 always being occupied with the repressor
How does lactose control the lac operon?
Lactose is converted to allolactose
Allolactose induces the lac operon by binding to the lac repressor and causing a conformational change
So lac repressor doesn’t constantly occupy the operator site
Induction of the lacZYA transcription
- RNA polymerase will bind but the lac repressor will prevent expression and will block RNA polymerase from initiating transcription.
- Lactose becomes available in the environment, it is taken up in the cell and converted to allolactose
- Allolactose binds to the lac repressor, it causes a conformational change in the lac repressor
- This reduces the affinity of the lac repressor for the lac operator, and RNA polymerase can initiate transcription.
- Transcription is said to be derepressed/induced.
How does glucose influence transcription of the lac operon?
Glucose levels influence levels of cAMP
cAMP is made from ATP by adenylate cyclase
Glucose metabolites prevent cAMP accumulation
As glucose level drops cAMP accumulates
cAMP binds to and activates CAP (CRP) Protein
• CAP = cAMP activator protein
• CRP = cAMP repressor protein
They bind to a site just upstream of the promotor
Activation of the lac operon by cAMP-CAP
Lac promoter has changes in both -35 and -10 consensus sequences so the CAP binding site compensates for the poor non-consensus promoters.
CAP loops contact the alpha subunit RNA polymerase directly (binds).
When CAP is bound (and there is no repressor) you get a 50 fold stimulation of transcription, which is a significant increase.
What happens when theres glucose but no lactose?
No transcription
Repressor blocks transcription
CAP doesn’t bind
What happens when theres no glucose and no lactose?
No transcription
High cAMP
Repressor blocks RNA polymerase
What happens when glucose and lactose are present?
Little transcription
Low cAMP
CAP does not activate
What happens when only lactose is present?
High transcription
No repressor
CAP activates
How is CAP involved in cholera?
Cholera – an acute, diarrheal illness that can result in severe dehydration & even death within a matter of hours
The organism (vibrio cholera) can exist both inside and outside human gut
• Colonisation of the human gut requires the activation of numerous genes
• Many of these genes are under control of the CAP activator
CRP mutants are also defective in intestinal colonisation
