Operons: Fine Control of Bacterial Transcription Chapter 7

Why is Operon system important? 3000 genes in E.coli – some products are in great demand Some genes are turned off – rarely needed Cannot leave all genes on all the time – takes energy for transcription and translation – drain cell of energy Scientists – use Operon system

The lac Operon The lac operon - first operon discovered Contains 3 genes coding for E. coli proteins that permit the bacteria to use the sugar lactose Galactoside permease (lacZ) - transports lactose into the cells b-galactosidase (lacY) cuts the lactose into galactose and glucose Galactoside transacetylase (lacA)- function is unclear In a flask containing glucose and lactose, the cells exhaust glucose and stop growing. For a short time it appears that it cannot adjust to lactose, but resumes growth after some time and turn on their lac operon to begin to accumulate their enzymes needed to metabolize lactose. Lactose is composed of two simple sugars galactose and glucose joined by beta galactosidic bond.

lac Operon All 3 genes are transcribed together producing 1 mRNA - a polycistronic message that starts from a single promoter Cistron or gene - has its own ribosome binding site A polycistronic message with information from more than one gene. Each cistron can be translated by separate ribosomes that bind independently of each other

Control of the lac Operon The lac operon is tightly controlled - using 2 types of control Negative control - like the brake of a car - binds the repressor to the operator Positive control – like accelerator pedal – removes repressor from operator Activator - additional positive factor - responds to low glucose by stimulating transcription of the lac operon The brake in negative control is a protein called lac repressor which keeps operon turned off or repressed as long as lactose is absent. Because it would be wasteful for the cell to produce enzymes needed to use an absent sugar. An additional factor activator is needed to activate operon. It responds to low glucose levels by stimulating transcription of lac operon, but high glucose levels keeps concentration of activator low so transcription of operon cannot be stimulated. The advantage of this positive control is that it keeps operon turned off when level of glucose is high as bacteria metabolize glucose more easily than lactose and it will be wasteful for them to activate the lac operon in presence of glucose

Negative Control of the lac Operon

Negative Control of the lac Operon Negative control - operon is turned on unless something turns it off and stops it The off-regulation is done by the lac repressor Product of the lacI gene Tetramer of 4 identical polypeptides Binds the operator just right of promoter Repressor is the protein that turns operon off. When repressor binds the operator, operon is repressed. The operator and promoter are contiguous. When repressor bind to operator then it prevents RNA polymerase from binding to the promoter and transcribing the operon. As long as no lactose is available, lac operon is repressed

lac repressor When repressor binds the operator - operon is repressed Operator and promoter are contiguous Repressor bound to operator prevents RNA polymerase from binding to the promoter As long as no lactose is available - lac operon is repressed

Inducer of lac operon The repressor is an allosteric protein Binding of one molecule (inducer – allolactose) to the protein (repressor) changes shape of a remote site on that protein Altering its interaction with a second molecule (operator) Inducer (one molecule) of lac operon binds the repressor causing the repressor to change conformation that favors release from the operator (the second molecule). The inducer is allolactose, an alternative form of lactose. This happens in absence of lactose

Mechanism Summary Two hypotheses of mechanism for repression of the lac operon RNA polymerase can bind to lac promoter in presence of repressor Repressor will inhibit transition from abortive transcription to processive transcription Repressor by binding to operator blocks access by the polymerase to adjacent promoter

Three lac operators Three lac operators The major lac operator lies adjacent to promoter Two auxiliary lac operators - one upstream and the other downstream All three operators are required for optimum repression The major operator produces only a modest amount of repression

Role in repression of transcription by the three operators

Catabolite repression When glucose is present - lac operon is in an inactive state Selection in favor of glucose attributed to role of a breakdown product – Catabolite Catabolite repression uses catabolite to repress the operon Selection in favor of glucose attributed to role of a breakdown product – Catabolite. The breakdown product is related to glucose or catabolite of glucose. This happens when both glucose and lactose are present. The bacteria will not use lactose until it metabolizes glucose

Positive Control of the lac Operon Positive control of lac operon by a substance sensing lack of glucose -activate lac promoter The conc. of nucleotide - cyclic-AMP - rises as the concentration of glucose drops

Catabolite Activator Protein cAMP added to E. coli can overcome catabolite repression of lac operon Positive controller of lac operon has 2 parts: cAMP + protein factor Protein factor is known as: Catabolite activator protein or CAP Cyclic-AMP receptor protein or CRP Gene encoding this protein is crp Addition of cAMP led to activation of the lac gene even in the presence of glucose

The Mechanism of CAP Action CAP-cAMP complex binds to the lac promoter Mutants whose lac gene is not stimulated by complex had the mutation in the lac promoter Mapping the DNA has shown that the activator-binding site lies just upstream of the promoter Binding of CAP and cAMP to the activator site helps RNA polymerase form an open promoter complex

CAP-cAMP complex The open promoter complex does not form even if RNA polymerase has bound to the DNA - till the CAP-cAMP complex is also bound CAP-cAMP recruits polymerase to the promoter in two steps Formation of the closed promoter complex Conversion of the closed promoter complex into the open promoter complex

CAP Binding sites for CAP in lac, gal and ara operons -contain the sequence TGTGA Binding of CAP-cAMP complex to DNA is tight CAP-cAMP activated operons have very weak promoters Their -35 boxes are quite unlike the consensus sequence Sequence conservation suggests an important role in CAP binding

CAP-cAMP Activation of lac Transcription The CAP-cAMP dimer binds to its target site on the DNA The aCTD (a-carboxy terminal domain) of polymerase interacts with a specific site on CAP Binding is strengthened between promoter and polymerase

ara Operon The AraC - ara control protein, acts as both a positive and negative regulator There are 3 binding sites Far upstream site, araO2 araO1 located between -106 and -144 araI is really 2 half-sites araI1 between -56 and -78 araI2 -35 to -51

araCBAD operon The ara operon is also called the araCBAD operon for its 4 genes Three genes, araB, A, and D, encode the arabinose metabolizing enzymes These are transcribed rightward from the promoter araPBAD Other gene, araC Encodes the control protein AraC Transcribed leftward from the araPc promoter

Control of the ara operon

AraC Control of the ara Operon In absence of arabinose - no araBAD products - AraC exerts negative control Binds to araO2 and araI1 Loops out the DNA in between Represses the operon Presence of arabinose - AraC changes conformation It can no longer bind to araO2 Occupies araI1 and araI2 instead Repression loop broken Operon is derepressed

Positive Control of the ara Operon Positive control is also mediated by CAP and cAMP The CAP-cAMP complex attaches to its binding site upstream of the araBAD promoter

The trp Operon The E. coli trp operon contains the genes for the enzymes - to make the amino acid tryptophan The trp operon codes for anabolic enzymes Anabolic enzymes are typically turned off by a high level of the substance produced Operon - negative control by a repressor - tryptophan levels are elevated trp operon also exhibits attenuation anabolic enzymes those that build up a substance. This operon is subject to negative control by a repressor when tryptophan levels are elevated

Tryptophan’s Role in Negative Control of the trp Operon Five genes code for the polypeptides in the enzymes of tryptophan synthesis – Chorismic acid The trp operator lies wholly within the trp promoter Presence of high tryptophan helps the trp repressor bind to its operator In lac operon, the promoter and operator precede the genes and same is true for trp operon. In trp operon the operator lies within the promoter. In positive control of lac operon, the cell senses the presence of lactose by appearance of tiny amounts of its rearranged product – allolactose. This causes the repressor to fall off lac operator and derepress the operon.

Negative control of trp operon Without tryptophan no trp repressor exists -inactive protein - aporepressor If aporepressor + tryptophan - changes conformation with high affinity for trp operator Combine aporepressor and tryptophan to have the trp repressor Tryptophan is a corepressor

Mechanism of Attenuation Attenuation imposes an extra level of control on an operon - more than just the repressor-operator system Operates by causing premature termination of the operon’s transcript when product is abundant The reason for premature termination is attenuator contains a transcription stop signal (terminator): an inverted repeat followed by a string of eightA-T pairs in a row. Because of inverted repeat the transcript of this region would tend to engage in intramolecular base pairing forming hairpin.

Defeating Attenuation Attenuation operates in the E. coli trp operon as long as tryptophan is plentiful If amino acid supply low - ribosomes stall at the tandem tryptophan codons in the trp leader trp leader being synthesized as stalling occurs - stalled ribosome will influence the way RNA folds Prevents formation of a hairpin

Overriding Attenuation

Riboswitches Small molecules can act directly on the 5’-UTRs of mRNAs to control their expression Regions of 5’-UTRs capable of altering their structures to control gene expression in response to ligand binding are called riboswitches Region that binds to the ligand is an aptamer

This project is funded by a grant awarded under the President’s Community Based Job Training Grant as implemented by the U.S. Department of Labor’s Employment and Training Administration (CB-15-162-06-60). NCC is an equal opportunity employer and does not discriminate on the following basis: against any individual in the United States, on the basis of race, color, religion, sex, national origin, age disability, political affiliation or belief; and against any beneficiary of programs financially assisted under Title I of the Workforce Investment Act of 1998 (WIA), on the basis of the beneficiary’s citizenship/status as a lawfully admitted immigrant authorized to work in the United States, or his or her participation in any WIA Title I-financially assisted program or activity.

Disclaimer This workforce solution was funded by a grant awarded under the President’s Community-Based Job Training Grants as implemented by the U.S. Department of Labor’s Employment and Training Administration.  The solution was created by the grantee and does not necessarily reflect the official position of the U.S. Department of Labor.  The Department of Labor makes no guarantees, warranties, or assurances of any kind, express or implied, with respect to such information, including any information on linked sites and including, but not limited to, accuracy of the information or its completeness, timeliness, usefulness, adequacy, continued availability, or ownership.  This solution is copyrighted by the institution that created it.  Internal use by an organization and/or personal use by an individual for non-commercial purposes is permissible.  All other uses require the prior authorization of the copyright owner.