Bacterial gene regulation Flashcards
molecular definitions of a gene?
1) the DNA sequence that is translated into a protein
2) the ORF and the control region of the gene including its promoter
dawkins definitions of a gene?
any portion of chromosomal material that potentially lasts for enough generations to serve as a unit of natural selection - Dawkins
flow of information in a cell?
DNA -> RNA -> protein
the central dogma describes…
…the flow of information in an organism
what do protein control points do?
removes rna / proteins which arent needed, for efficiency
in bacteria, transcription ansd translation is…
…coupled
where is bacteria in dna?
chromosomal DNA is free in the cytoplasm
because the chromosomal DNA is free in the cytoplasm translation can begin as soon as enough transcript is available for ribosome binding – this enables…
… gene regulation to be fast
the expression of genes is regulated at several levels which are?
1) transcriptional control
2) post-transcriptional control
3) translational control
4) post-translational control
Function of transcriptional control?
control of production of the mRNA transcript – requires promoters/operators (DNA) and activators/repressors (proteins) e.g. lac operon
Whats the function of post-transcriptional control?
mRNA stability – how long is the mRNA available for ribosomes to translate? e.g. Crp-cAMP control by RNaseL
whats the function of translational control?
inhibition of ribosome binding – e.g. threonyl tRNA synthetase (ThrS) binds to thrS mRNA inhibiting ribosome binding
Whats the function of post translational control?
activation or inactivation of an already translated protein
a) protein stability/degradation rates
b) post-translational modification – addition of phosphate groups (phosphorylation)
Whats the most important level of gene expression in bacteria?
In bactiera, the mos timportant level of control is transcriptional control
How many regions in each gene?
3
What are the three regions of a gene?
1) 5’ promoter, attracts RNA polymerase
2) transcribed sequence (transcript) or RNA coding sequence
3) 3’ terminator, signals the stop point
Whats the function of the RNA-coding sequence?
rna coding sequence starting with aug and ending with stop codon
Function of the +1 on a gene?
where transcription starts.
Whats the terminator of a gene?
stops transcribing and moves off gene.
Function of promoter?
The function of a promoter in transcription is to control when and where a gene is expressed in an organism
What is RNA polymerase made up of?
RNA polymerase made up of subunits, transcribes all genes.
unlike in eukaryotes only one bacterial RNA polymerase transcribes…
… all genes – coding genes, rRNA and tRNA genes
What does RNA polymerase enzyme consist of?
the enzyme consists of the core alpha and beta subunits
What are the core alpha and beta subunits of RNA polymerase involved in?
formation of RNA via phosphodiester bonds adding nucleotides to a growing RNA chain.
The rna polymerase complex is unable to…
… bind to DNA itself in order to initiate transcription
how does the rna polymerase complex bind to dna?
binding takes place via sigma (σ) factors
differential binding of sigma factors represents the most…
… general part of gene regulation in bacteria
control of transcription in bacteria requires…
… the presence of a promoter just upstream of the transcription initiation site
control of transcription in bacteria requires the presence of a promoter just upstream of the transcription initiation site – conservation occurs in…
… the promoter as these are DNA sequences that interact with proteins that are required to initiate transcription – sigma factors
Whats a sigma factor?
proteins that are required to initiate transcription
What are sigma factors required for?
transcription
different sigma factors control…
… different sets of genes.
How many sigma factors in E. coli?
in E. coli there are 7 different sigma factors each responsible for transcription initiation of different regulons
How does a sigma factor work?
1) Sigma factor recognises and binds to -35 and -10 sequences
2) Then sigma factor changes its shape so it can bind to rna polymerase
3) So then it known where to bind on a promoter and start transcription.
sigma factors bind to …
… DNA
the grooves within a double stranded DNA molecule are able to …
… interact with amino acids on the surface of a protein due to H-bonds and Van der Waals forces
the grooves within a double stranded DNA molecule are able to interact with amino acids on the surface of a protein due to H-bonds and Van der Waals forces
this provides the opportunity for proteins to …
… interact and importantly the interactions are sequence specific – hence the core promoter sequence being conserved!
Sigma factor changing shape of protein allows …
… the dna to interaction with it
Sigma factor changing shape of protein allows the dna to interaction with it. Because …
… Because the dna fits to the changed shape of the protein.
Sigma factor knows where to go
sigma factors are released on …
… initiation
initiation of transcription causes …
… the sigma factor to be released and RNA polymerase processes along the template strand.
Bound sigma factor recruiting …
… rna polymerase to the correct position
Rna polymerase opens …
… dna double strand
1) Bound sigma factor recruiting rna polymerase to the correct position.
2) Rna polymerase opens dna double strand
3) After, sigma factor …
… moves off and is used elsewhere. Because rna polym has begun to do its job of unwiinidng the dna double stran in 5 to 3 direction
the template strand enters through …
.. a part of the holoenzyme called the DS channel and exits through the T channel
the growing mRNA molecule exits through …
… the RNA channel
nucleotides enter through the…
… NT channel
synthesis of a mRNA transcript proceeds much as …
… DNA polymerisation
What does RNA polymerase cover?
the enzyme covers an open region of the template strand (the transcription bubble)
the actual single stranded DNA template is around…
… 12 bases
where are new bases added to the growing mRNA?
bases are added to the 3’ OH group on the growing mRNA strand by complementary base pairing (A to T, C to G, G to C and U to A)
mRNA synthesis is …
synthesis is discontinuous – has pauses this is important in termination.
synthesis is discontinuous
What does this mean?
has pauses – stopping and starting
What is the stopping and starting important for?
termination. Allows decisions to be made as to whether to keep transcribing or terminating transcription.
the pausing allows RNA polymerase to make a…
… ‘choice’ between continuing transcription or terminating transcription
the choice is determined by…
… which ‘decision’ is the most energetically favourable
RNA molecules also base pair – at an intrinsic terminator…
… the RNA/RNA pairing is more energetically favourable than RNA/DNA pairing.
the number of RNA/DNA base pairs in the transcription bubble decreases and …
… reduces the stability of the complex and dissociation occurs
Whats the mal operon responsible for?
responsible for metabolising maltose
in the mal operon which is responsible for metabolising maltose control is by …
… activation rather than derepression (as in lac)
What does maltose create?
Mal operon creates enzymes that breakdown maltose into glucose.
Bacteria only transcribe mal opern when …
… maltose is present
Bacteria only transcribe mal opern when maltose is present
Its saving …
… energy by only making them when needed. If mal opern was transcribed all the time this would be a waste of time.
What does maltose bind to?
maltose binds to an activator protein when it is present in the cell
maltose binds to an activator protein when it is present in the cell
this causes …
… a conformational change in the activator
maltose binds to an activator protein when it is present in the cell
this causes a conformational change in the activator
the activator binds …
… to the promoter
maltose binds to an activator protein when it is present in the cell
this causes a conformational change in the activator
the activator binds the promoter
transcription is…
… initiated
What is the arg operon is controlled by?
the arg operon is controlled by repression of transcription
the arg operon is controlled by repression of transcription – in this case the presence of the product of the operon is …
… arginine
Whats the co-repressor in the arg operon?
arginine itself acts as a co-repressor
arginine itself acts as a co-repressor – binding of arginine to the repressor causes…
… conformational shift and it binds to the operator
Effect of adding arginine to a bacterial culture on its growth and synthesis of proteins?
the addition of arginine to a bacterial culture has no effect on total growth and synthesis of proteins
what does arginine specifically do?
it specifically shuts down the genes involved in arginine biosynthesis
the regulation of the arg operon is…
… specific - other operons are unaffected.
what are there no run of after hairpin?
no run of As
What does Rho protein follow?
Rho protein follows the RNA polymerase – attaches to the transcript
if the RNA polymerase stalls then …
…Rho catches up and unwinds the RNA/DNA strand in the transcription bubble
if the RNA polymerase stalls then Rho catches up and unwinds the RNA/DNA strand in the transcription bubble.
What happens then?
transcription stops
if the RNA polymerase stalls then Rho catches up and unwinds the RNA/DNA strand in the transcription bubble
transcription stops
What are these kinds of terminators called?
Rho-dependent terminators
what is used in the regulation of termination?
antiterminators
What do antiterminators bind to?
antiterminator proteins bind to positions on the DNA near the beginning of an operon.
slide 19
Whats trp stand for?
tryptophan
How many levels of contorl in the trp operon?
two levels of control
Describe the two levels of control in the trp operon?
controlled by a repressor protein with binds to the operator when tryptophan associates with the repressor (ie. when there is a lot of tryptophan in the cell and it doesnt need to be made.
what does binding of tryptophan to the repressor cause?
a conformational change and DNA binding sites on the protein are exposed
the trp operon is down-regulated to a large extent by …
… the repressor but the down-regulation is modified even further by attenuation.
Trp binds to and changes shape of…
… the active repressor
Trp binds to and changes shape of the active repressor
Active repressor now …
… fits with operator. They bind. Turns the genes off.
because both the ribosome and the RNA polymerase are attached to the mRNA transcript at the same time this provides …
… an opportunity for control.
because both the ribosome and the RNA polymerase are attached to the mRNA transcript at the same time this provides an opportunity for control
this type of control is …
… attenuation
What is attenuation particularly associated with?
particularly associated with operons that control amino acid biosynthesis
What does trp operon control?
the trp operon controls the biosynthesis of tryptophan
Describe trp operon and biosynthesis of tryptophan?
if the bacterial cell has enough tryptophan it makes sense to turn production of these biosynthetic enzymes off
Why might the ribosome stall?
when there is little tryptophan.
What happens when the ribosome stalls when there is little tryptophan?
the large hairpin or non-terminator loop forms
what happens to the ribosome if there is an excess of tryptophan?
if there is an excess of tryptophan then the ribosome keeps pace with the RNA polymerase
the large hairpin does not form and instead the short terminator hairpin forms
if there is an excess of tryptophan then the ribosome keeps pace with the RNA polymerase
the large hairpin…
… does not form and instead the short terminator hairpin forms
if there is an excess of tryptophan then the ribosome keeps pace with the RNA polymerase
the large hairpin does not form and instead the short terminator hairpin forms
what does this cause the RNA polymerase to do?
this causes the RNA polymerase to terminate transcription
Why does the ribosome stall when tryptophan is scarce?
the ribosome stalls because there is not enough tryptophan to make the leader peptide quickly
When tryptophan stalls, what forms?
the large hairpin loop forms – transcription continues
What happens to transcription when the ribosome stalls and the large hairpin loop forms?
transcription continues.
What happens to the ribosome when tryptophan is abbundant?
the ribosome proceeds quickly and keeps pace with RNA polymerase – the terminator loop forms – transcription stops
where are leader peptides found?
seen at the beginning of operons for synthesis of several amino acids
what does the leader peptide contain ?
contains an excess of the operon’s end product
