Gene regulation in bacteria 2 - operons

Lac operon

- Comprised of 3 genes: lac Z, lac Y and lac A Lac Z is the gene for beta-galactosidase Lac Y is for lactose permease (transporter that brings lactose into the cell) Lac A is for thiogalactoside transacetylase (helps remove toxic metabolites e.g. thiogalactosides, transported out of the cell by the Lac Y protein) (still not quite sure what this protein does) - RNA polymerase binds to the promoter (P) sequence. - The operator (O) region that helps in the decision whether the gene is transcribed or not - The Lac I gene is not part of the operon (has its own promoter), codes for the repressor protein that is involved in the decision of whether the lac operon is transcribed or not. - The lac I gene is transcribed from its own promoter and is constitutively expressed.

Phophate operon

- A 2 component regulatory system containing an activator (sensor) protein (PhoR) and a regulator protein (PhoB). When [phosphate] drops, the cell's survival depends upon proteins that will increase [phosphate]. Hence, transcription must be activated when [phosphate] drops too low. - The PhoR protein acts as the activator (sensor). This transmembrane protein has a domain inside the cytoplasm and a domain which stretches across the membrane into the periplasm. - The domain in the periplasm is normally bound to a phosphate (when the concentration is normal). - When the phosphate concentration is insufficient, the protein undergoes a conformational change which exposes histidine. This histidine is then phosphorylated by ATP to produce the activated PhoR. - The phosphate group from the PhoR is used to phosphorylate phoB (at the aspartate) which becomes activated. This enables transcription as PhoB binds to DNA and helps (increases affinity) RNA Polymerase bind to the promoter. Examples of genes activated include: phoA - alkaline phosphatase phoS - phosphate binding protein phoE - porin ugpB - phosphate transporter

Attenuation

- A process in which the transcription of the mRNA molecule is stopped after the attenuator sequence (161bp), in the circumstance that tryptophan levels are high. The full mRNA for trp operon is 7000 bases - Attenuation depends on the folding pattern of the leader mRNA and extent to which ribosome has translated the leader sequence. There are 4 regions within the leader mRNA which have complimentarity. When these regions base pair together, they form a hairpin loop structure which terminates transcription. - If 1 base pairs to 2, there is no transcription (no ribosome begins translation) - If 3 base pairs to 4, a termination hairpin forms and there is attenuation . (Happens when there is high levels of tryptophan. - If 2 base pairs to 3, 3 cannot base pair to 4 and therefore transcription is not terminated. (Happens during low levels of tryptophan as there are 2 UGG codons for trp embedded within sequence 1. This means that the ribosome is stalled at sequence 1 when tRNA is trying to find tryptophan to bind to in order to elongate the AA chain. This effectively stops sequence 1 from pairing to 2.)

Polycistronic mRNA

- An mRNA that has been transcribed from more than one gene. (genes used to be called cistrons)

Control of gene expression

- Cell does not transcribe all its genes for no purpose - Spectrum of proteins present depends on the nutrients that are available to the cell - Some proteins are always present in constant amount (house keeping proteins - necessary to keep the cell alive) - Other proteins vary in amount - the concentration is controlled by gene expression e.g. sugar metabolism, amino acid synthesis

Positive control of lac operon

- Glucose inhibits transcription of lac operon, whilst lactose is present (no lactose=no transcription anyways). - The cell will always use the most efficient nutrient that is available to it. Therefore, this mechanism has evolved in order to allow this (glucose is more efficient than lactose as glucose metabolism does not need new proteins). - The concentration of cAMP (a metabolite of glucose) is critical in the decision making of whether the lac operon is transcribed. This is called 'catabolite repression'. - Concentration of glucose is inversely proportional to conc of cAMP. - When glucose enters the cell, an enzyme phosphorylates it into glucose-6-phosphate (g6p). This is done because g6p cannot move out of the cell. In this state, adenylyl cyclase is inactive. - In the absence of glucose, adenylyl cyclase is phosphorylated and becomes active. This allows it to facilitate the conversion of ATP into cAMP. - cAMP binds to the CAP (catabolite activator protein) to create the CAP-cAMP complex. This CAP complex binds to the RNA polymerase and allows it to effectively bind to the promoter region (which otherwise would not be possible due to the weak promoter of the lac operon) and transcribe the lac operon efficiently. This cannot happen if glucose is present in the cell which is why glucose inhibits the transcription of the lac operon.

Lac operon - repressed (normal) state

- In the normal state of the lac operon, the repressor protein from Lac I binds to the operator region and prevents RNA polymerase from binding, hence prevents transcription of the lac operon.

Genetic analysis of lac operon regulation (1940s)

- Jacob and Monod created partial diploid cells. They had a normal chromosome and included an F plasmid which also contained the lac operon (they are making a partial diploid with respect to the lac operon). - The aim of the experiment was to understand if the chromosome and the plasmid could talk to one another. - The chromosome had a mutation in the lac I gene (lac I-), such that the repressor cannot bind to the operator. Hence, the operator is still on even in absence of lactose. - The F plasmid contained the normal lac I gene. The question was: Can the lac I+ on the F plasmid complement the lac I- mutation on the chromosome and restore regulation of the lac operon? - Yes. They had shown that some information had crossed from the plasmid into the chromosome and restored the regulation. They concluded that there was a protein in the cell that was moving and binding to restore regulation. Ingenious how they found the answer. They did another experiment to examine the DNA and learn more about the regulation of the operon. They created a mutation in the operator sequence (Oc where c=constitutive) such that the repressor can't bind. The question here was: Can the Oc mutation on the chromosome affect O+ on plasmid. To be able to do this they needed to create the Z- mutation on the chromosome so that it could not produce the enzyme. This ensured that the only b-galactosidase produced was from the plasmid (which they wanted to monitor). - They found that the lac operon was still induced in the presence of IPTG (an inducer, an analogue of allolactose) which meant that the info on the chromosome did not transfer to the plasmid. They concluded that the a DNA sequence as is involved in the regulation as DNA does not move in the cell.

Lac operon - activated state

- The repressor protein has a binding site for both DNA and the lactose molecule. However, these are mutually exclusive (when it binds to one it cannot bind to the other). Therefore, when there is lactose, the repressor binds to it and there is a conformation change which doesn't allow it to bind to the promoter. Hence, transcription is allowed.

trp operon

- The trp operon contains 5 genes: trpE,D,C,B and A (The overall affect of the 5 genes is to synthesise tryptophan); an operator and promoter region; and a leader sequence (trp L) which contains a small sequence called the attenuation sequence (trp a) (involved in an additional regulatory process). - trp R (has its own promoter) codes for an aporepressor (a repressor that has to bind with a co-repressor in order to inhibit transcription) which is regulating the trp operon. Therefore, when tryptophan is present (co-pressor), the operon is repressed and vice versa.

Repressible operon

- Transcription is switched off when sufficient product is in the cell e.g. Tryptophan operon (trp operon)

2 anomalies with the lac operon

- Transport of the inducer (lactose) requires lactose permease (lac Y) - The 'true' inducer is not lactose but rather allolactose (lactose undergoes isomerisation to form allolactose, facilitated by B-galactosidase) The solution to this paradox is that the binding of the repressor is never infinitely strong (dynamic) and it will drop off approximately once every cell generation (there is always a very low level of lac transcription).

tRNAs and rRNAs

- tRNA & rRNA are very stable (energetically favourable) whereas mRNA is not (allows regulation of transcription). - tRNAs are transcribed with 5' and 3' flanking sequences which are removed to produce mature tRNAs. - Ribonuclease P (RNase P) is an endonuclease which cleaves phosphodiester bonds internally - Ribonuclease D (RNase D) is an exonuclease which cleaves phosphodiester bonds one at a time until it reaches the CCA sequence which is at the end of all tRNAs. - E.coli has 7 rRNA operons. - They are all made in equal amounts. - RNase III makes staggered cuts in the stem loop structure of rRNA.

Operon

A cluster of genes coding for the same metabolic pathway with a single promoter. - It is transcribed into 1 mRNA molecule (polycistronic mRNA). - Makes sense to have 1 promoter controlling all genes in a particular pathway if the organism only has a short time to live as all genes in the pathway can be neatly transcribed at the same time.

Control of operons in bacteria

Bacteria obey a simple rule - they turn on transcription when it is needed and off when it isn't - they allow RNA synthesis or prevented. 2 main types of operons: Inducible - transcription is turned on Repressible - transcription is turned off Control of every operon involves 2 key features: small molecule + regulatory protein

Inducible operon

Normally involved in a catabolic process e.g. sugar breakdown. Small molecule = substrate ( or closely related molecule) to first enzyme in the metabolic pathway. e.g. lactose operon (lac operon) - 1st enzyme in the pathway is Beta-galactosidase & small molecule = glucose. - Beta-galactosidase cleaves the beta-galactoside linkage in the lactose molecule to produce glucose and galactose. - When lactose is added to a bacterial culture, there is a massive increase in the amount of beta-galactosidase in a short amount of time and a sharp decrease after the lactose is removed. This phenomenon displays the regulatory nature of the operon.

CAP increases affinity of RNA polymerase for the weak lac promoter

The alpha subunit of the RNA polymerase has 2 domains that play a significant role in this: alpha CTD (carboxy-terminal domain) & alpha NTD (amino-terminal domain). - The alpha CTD makes contact with the CAP-cAMP complex and the alpha NTD makes contact with the other RNA pol subunits. - The alpha subunits are important in stabilising the interaction between RNA pol and the CAP-cAMP complex on the DNA. - The CAP-cAMP complex binds to the DNA and bends it by slightly more than 90 degrees which enables RNA pol to gain increased traction on the DNA (increases affinity for RNA pol to lac operator).

lac operator

The lac operator sequence is a near perfect inverted repeat (the repeated motifs in DNA indicate that the sequence is recognised by protein - called 'half-sites'). - The principle operator O1 contains 2 'half-sites' which binds 2 repressor molecules. - There also exists an auxiliary operator O3 - The active repressor from the lac I gene is a tetramer as 2 molecules bind to each operator and there are 2 promoters - so it binds as a tetramer. This formation effectively blocks RNA polymerase. - For transcription to take place, molecules of lactose has to bind to each of the 4 subunits. Transcription of the lac operon is described as being negatively regulated as the operon is off when the repressor is bound.

Quorum sensing in vibrio fischeri

Vibrio fischeri when present in large numbers cause fish and squid to illuminate. However, when they are in small numbers/alone they do not. - Each bacterium produces the signalling molecule:VAI (Vibrio fischeri autoinducer) which increases transcription of the lux operon. - The lux operon consists of the promoter & operator, lux I (codes for the enzyme for autoinducer synthesis 'VAI synthesase'), lux A & B (codes for luciferase - enzyme which produces light) and lux C,D & E (codes for luciferin - substrate for luciferase). - The luxR (has its own promoter) codes for the transcriptional activator, which binds to the VAI (produced by VAI synthesase) to create the regulator complex. This complex binds to DNA and assists RNA polymerase in binding to the promoter. This increases the transcription of the other genes. - Note that VAI must already exist at a low level in the gene for this to be possible.

Equation for bioluminescence

reduced flavin + oxygen + luciferin > oxidised flavin + oxidised luciferin + blue-green light