Gene regulation in bacteria II
What does the trp operon consist of?
-5 protein coding genes, whose products are involved in the synthesis of Trp -These genes are found in the order (downstream from the promoter): trpE, trpD, trpC, trpB, trpA -A promoter sequence -An operator, which is found downstream of the promoter -trpL - this is found between the operator and trpE, and encodes a small 'leader sequence' which is not involved in trp production but is involved in regulation of transcription of the protein-coding genes
Explain how glucose is an inhibitor of lac operon transcription.
-Adenylyl cyclase, the enzyme that produces cAMP from ATP, effectively acts as an indirect source of phosphate groups for glucose-6-phosphate -When glucose is phosphorylated, adenylyl cyclase loses its phosphate group -This inactivates adenylyl cyclase, preventing the production of cAMP -cAMP is a positive regulator of the lac operon, therefore loss of cAMP results in loss of lac operon transcription -Even if lactose is present, presence of glucose usually leads to repression of the lac operon -However, some transcription may occur if glucose levels are low enough such that there is still some cAMP left
What is a positive regulator of the lac operon?
-Binding of cAMP to the promoter is essential for transcription of the lac operon to occur - cAMP is a positive regulator of transcription -cAMP binds to catabolite activator protein (CAP) -The cAMP-CAP complex is able to bend DNA at approximately a right angle -It is believed that this exposes the lac promoter to RNA polymerase so that RNA polymerase is able to bind with sufficient strength
How is the rRNA precursor produced? How is it then processed?
-E.coli has seven ribosomal rRNA operons (rrn operons) -Each operon encodes three rRNAs and two tRNAs in the order: 16S, tRNA, 23S, 5S, tRNA -The primary transcript is cleaved to separate the elements of the primary transcript -tRNA molecules are processed as usual using RNaseP and RNaseD -The 23S and 16S rRNA molecules are still too large -Both transcripts contain regions of complementarity which hybridise to one another -This forms a double-stranded stem which is the target for RNaseIII -RNaseIII makes a double-stranded cut which releases the excess RNA from the mature rRNA molecule
How do bacteria arrange genes?
-Genes that are involved in the same metabolic pathway are often clustered into an operon -The operon has a single promoter, so the genes are always transcribed together into one mRNA molecule
How does beta-galactosidase act on lactose?
-It breaks the beta galactosidase link which connects the two sugars in lactose -This produces one glucose molecule and one galactose molecule
Describe the lac operator.
-It is a near-perfect inverted repeat and can thus be divided into two half-sites -There are actually three operators, including the primary operator O1 and the auxiliary operator O3 -O1 is found upstream of the promoter and binds two repressor molecules, one to each half-site -O3 is found downstream of the promoter and also binds two repressor molecules, one to each half-site -The four repressor molecules interact and form a tetramer - this bends the promoter region into a loop
How was the function of the lac operator region discovered?
-It was originally unknown whether the operator region was protein-coding or not -Partial diploid cells were created, with an O^C mutation and a lacZ- mutation on the bacterial chromosome and fully functional genes (O+) on an F plasmid -The lacZ- mutation was added so that products of F-plasmid lac operon and chromosomal lac operon could be distinguised -Cells without the F plasmid constitutively expressed the lac operon due to the O^C mutation; however, a non-functional form of beta-galactosidase was expressed due to the lacZ- mutation -IPTG was added to act as a repressor of the lac repressor (instead of lactose) -In the presence of the inducer, beta-galactosidase was produced from both operons so both functional and non-functional forms were found -In the absence of the inducer, only non-functional beta-galactosidase was produced - therefore only the chromosomal lac operon was activated -At this stage it was known that the lac operon binds to DNA to inhibit transcription of the lac operon; this experiment shows that the lac repressor binds to the operator region
What happens when lactose is added to the lac operon?
-Lactose binds to the lac repressor -This displaces the lac repressor from the DNA so that RNA polymerase is able to bind and transcribe the operon
Describe the structure of trpL?
-Leader mRNA (encoded by trpL) contains four sequences - 1, 2, 3 and 4 - which influence attenuation -Region 1 can base pair with region 2, region 2 can base pair with region 3, and region 3 can base pair with region 4 (regions that base pair with one another possess some complementarity) -Region 1 of the leader sequence encodes the 'leader peptide' which contains 2 Trp residues -The leader peptide has its own stop codon which is found in region 2
What does a two-component regulatory system rely on?
-Activator (sensor) protein - this detects changes in the cellular environment -Regulator protein - this responds to changes in the environment by altering transcription levels
Why has the lac operon taught us so much about operons?
Because it is the first operon whose mechanism was understood.
What are tRNAs and rRNAs derived from?
Long, short-lived precursor molecules.
How is transcription from the lac operon regulated? (general)
Negatively; the operon is switched off when the regulatory protein is bound.
In what order are the elements of the lac operon found?
Operator - promoter - lacZ - lacY - lacA
What rule do bacteria obey?
Transcription is turned on when it is needed, and turned off when it is not needed.
How was it discovered that the lacI gene is involved in negative regulation of the lac operon?
-Partial diploid cells were created, consisting of the E.coli chromosome and an F plasmid containing part of the E.coli chromosome -The full chromosome contained a lacI mutation termed lacI-; this mutation prevented the repressor from binding to the operator and thus the lac operon was constitutively expressed -The F-plasmid contained a fully functional lacI gene (lacI+) -It was found that the lacI+ gene complemented the lacI- gene - when the F plasmid was added, transcription of the lac operon was repressed -Therefore the product of the lacI gene must be involved in negative regulation of the lac operon
Give some examples of genes that are upregulated by PhoB.
-PhoA: alkaline phosphatase, liberates phosphate groups from a variety of biological molecules -PhoS: phosphate binding protein -PhoE: a porin which spans the cell wall and acts as a channel through which phosphate can enter -UgpB: phosphate transporter
What are housekeeping proteins?
-Proteins that are always present at constant levels -These proteins are constantly required for cell survival and are constitutively expressed (i.e. not regulated)
What are the advantages of operons?
-Saves space inside the bacterial cell -Increases efficiency of gene expression
How does attenuation work when Trp concentration is low?
-Transcription of the leader sequence occurs and the leader peptide begins translation whilst the rest of the sequence is being transcribed -Because of the lack of Trp in the cell, translation stops when the ribosome reaches the Trp codons in the leader peptide -The ribosome is held in region 1, which blocks the binding of region 1 to region 2 -Region 2 is able to bind to region 3, forming a hairpin loop -This hairpin loop is not a termination loop - it does not result in rho-dependent termination - so transcription of the Trp operon continues
How does attenuation work when trp concentration is high?
-Transcription of the leader sequence occurs and the leader peptide begins translation whilst the rest of the sequence is being transcribed -Translation stops at the stop codon for the leader peptide in region 2 - the ribosome cannot move forwards -Region 2 is thus blocked from binding to region 3 -Regions 3 and 4 are transcribed but not translated - they are thus able to hybridise to form a hairpin loop -The double-stranded stem of the hairpin loop is GC rich; rho-independent termination occurs and expression of the trp operon does not occur
How is the trp operon repressed in the presence of Trp?
-Trp binds to the trpR aporepressor - this causes a conformational change in the aporepressor which allows it to become a fully functional repressor -The repressor binds to the operator and thus partially blocks the promoter sequence -RNA polymerase cannot bind to the promoter sequence and trp operon transcription is repressed
How does bioluminescence occur in V.fischeri?
-V.fischeri possess the lux operon -Each bacterium also produces the signalling molecule Vibrio fischeri autoinducer (VAI), which increases transcription of the lux operon -Separate to the lux operon is the luxR gene, which has its own promoter and encodes LuxR protein -VAI binds to LuxR; this enables LuxR to bind to the operator region of the lux operon -Binding of LuxR to the lux operon increases RNA polymerase affinity for the lux promoter; transcription of the lux operon is increased -VAI synthetase is expressed which results in the production of VAI and further upregulation of the lux operon -Luciferase and luciferin are also expressed; luciferin is the substrate of luciferase and interaction between the two results in production of light
How is the tRNA precursor processed to produce mature tRNA?
-tRNA genes are arranged in an operon, producing a tRNA precursor containing 3 tRNA molecules, 2 sequences separating the tRNA molecules, and a 5' and 3' flanking sequence -Cleavage of the 5' flanking sequences is carried out by the endonuclease RNaseP; this leaves three tRNA precursors each with a 3' flanking sequence, as well as one piece of excess DNA -The exonuclease RNaseD binds to the 3' end of each tRNA precursor and sequentially cleaves phosphodiester bonds until it reaches a CCAOH3' (i.e. CCA, 3' hydroxyl) signal at the boundary between the 3' flanking sequence and tRNA
What is a quorum?
A minimum population.
What is typically the molecule involved in regulation of an inducible operon?
A substrate of the first enzyme involved in the catabolic pathway that the operon is involved in, or a molecule closely related to the substrate.
What is the true inducer of the lac operon? Explain the effects of this.
Allolactose, NOT lactose. -Allolactose is not the substrate of the first enzyme in the lactose breakdown pathway (beta-galactosidase), but is in fact the product -Paradoxically, activation of the lac operon is required to produce beta-galactosidase in order to produce allolactose and activate the lac operon -The lac repressor does not have infinite binding strength to the lac operator, so sometimes dissociates from the operator even in the absence of lactose -There is therefore always a low level of lac transcription, which provides enough beta galactosidase to produce small amounts of allolactose and truly activate the lac operon
How many proteins can E.coli code for?
Around 4000.
What do inducible operons tend to be involved in?
Catabolic pathways (i.e. the breakdown of a substrate to produce something that the cell needs)
What is the function of the lac operon?
Encodes proteins involved in the breakdown of lactose
What happens as V.fischeri levels increase inside the host?
Epithelial cells of the light producing organ expand.
What is a disadvantage of operons?
Gene expression is made somewhat inflexible.
What genes does the lac operon encode?
In downstream order from the promoter: -lacZ: this encodes beta galactosidase -lacY: this encodes lactose permease, which facilitates the transport of lactose into the cell and toxic metabolites out of the cell -lacA: this encodes thiogalactoside transacetylase, which helps to remove toxic metabolites
What are the two types of operon? Give examples.
Inducible - gene expression is turned on when a chemical is added (e.g. the lac operon; the chemical is lactose) Repressible - gene expression is turned off when a chemical is added (e.g. the trp operon; the chemical is tryptophan)
What is an important feature of the lacI gene?
It is constitutively expressed.
What is a feature of the mRNA transcript in prokaryotes that is not found in eukaryotes?
Multiple AUG (start) codons, and multiple stop codons. During translation, a ribosome attaches to each start codon so translation of all the genes occurs simultaneously.
What is the order of elements on the lux operon? What do the protein-coding genes encode?
Operator - promoter - luxI - luxC - luxD - luxA - luxB - luxE luxA and luxB encode luciferase, an enzyme which allows light to be produced luxC, luxD and luxE encode luciferin, the substrate for luciferase luxI encodes VAI synthetase, which produces Vibrio fischeri autoinducer (VAI)
Elements of the trp operon are found in what order?
Promoter - operator - trpL - trpE - trpD - trpC - trpB - trpA
What is the name of the phenomenon in which fish and squid are able to fluoresce? Which bacterium is involved in this? What is a feature of the phenomenon? (general)
Quorum sensing Vibrio fischeri -Small numbers of V.fischeri do not fluoresce - a quorum is required for fluorescence
What is an example of a two-component regulatory system? Explain how this works
REGULATION OF PHOSPHATE LEVELS -Phosphate is essential for cell survival -When [phosphate] drops, cell survival depends on the presence of proteins that will increase the levels of [phosphate] -Transcription must therefore be activated -The activator protein is PhoR, which spans the inner membrane of Gram-negative bacteria -PhoR possesses a phosphate binding site in the periplasmic space; phosphate is bound to PhoR when phosphate levels are sufficient -When phosphate levels are low, phosphate dissociates from PhoR -This causes a conformational change in PhoR which causes a previously embedded His residue to be exposed on the cytoplasmic side of the protein -The His residue is phosphorylated by ATP hydrolysis and PhoR is thus activated -PhoB is the regulator protein; it is a soluble transcription and is found in the cytoplasm -Activation of PhoR allows phosphorylation of an aspartate (D) residue on PhoB -This causes a conformational change in PhoB which allows it to bind to DNA and upregulate the transcription of many proteins required for phosphate production and uptake
Why do fish and squid host V.fischeri?
Symbiotic relationship: -The fish/squid provides a safe environment for V.fischeri -It is believed that the luminescence provided from V.fischeri allows the host to mimic moonlight and thus attract prey
What is found upstream of the lac operon?
The lacI gene, which has its own promoter. This encodes the lac repressor, which binds to the operator region of the lac operon in the absence of lactose. This causes a change in DNA structure which prevents RNA polymerase from binding to the operon.
What is found upstream of the trp operon?
The trpR gene, which has its own promoter and is constitutively expressed. trpR encodes an aporepressor - an immature repressor which needs to bind a co-repressor (in this case, Trp) in order to become a fully functional repressor.
How do tRNA and rRNA differ from mRNA?
They are not translated and they are much more stable. Because of their increased stability, they make up 98% of cellular RNA despite only making up 1% of the RNA-encoding genome.
What is polycistronic mRNA?
mRNA that encodes multiple genes.