Biology CH 18: Control of gene expression in bacteria

What are the three hypotheses central to Jacob-Monod model of the lac operon regulation?

1) The lacZ, Y, and A genes are adjacent and are transcribed into one mRNA initiated from the single promoter of the lac operon. As a result, the expression of the three genes is coordinated. 2) the repressor is a protein endoced by lacI that binds to DNA & prevents transcription of lacZ, Y, and A. LacI is expressed constitutively and that the repressor binds to the operator. 3) The inducer (lactose) interacts directly w/ the repressor by binding to it. As a result, the repressor changes shape in a way that causes it drop off the DNA strand. When the inducer binds to repressor, negative control ends & transcription can proceed.

lacA

A gene that is tightly linked to lacY and Z and part of the same operon. The enzyme's function is protective in nature. It catalyzes rxns that allow certain sugar types to be exported from the cell when they're too abundant.

cyclic AMP (cAMP)

A regulatory molecule that binds to the catabolite activator protein (CAP) and changes CAP's shape. During positive control, CAP-cAMP complex binds to the CAP binding site. As long as a repressor isn't bound to the operator, the complex increases efficiency and frequency of transcription and RNA polymerase bound tightly to promoter. If no cAMP is bound to CAP, then CAP has a conformation that does not allow binding to the CAP site. RNA polymerase is bounded loosely to promoter and transcription in infrequent.

catabolite activator protein (CAP)

A regulatory protein which binds to the CAP binding site on the DNA sequence. Triggers transcription of the lac operon.

What happens when a repressor is present and lactose absent?

A repressor synthesized from normal lacI gene binds to DNA ahead of the RNA polymerase that is bound to a promoter. Transcription is blocked and no lacZ or Y is expressed.

genetic screen

A technique that allows researchers to identify individuals with a particular type of mutation.

operon

A term for a set of coordinately regulated bacterial genes that are transcribed together into one mRNA. The group of genes involved in lactose metabolism was termed the lac operon.

Which of these three forms of control occur in bacteria?

All three: transcriptional, translational, and post-translational. Transcriptional is important due to its efficiency-- although slow, it saves the most energy for the cell, because it stops the process at the earliest point possible. Translational is advantageous because it allows a cell to make rapid changes in its array of proteins. Post-translational is significant because it provides the most rapid response. However, it is energetically expensive.

translational control

Allows the cell to control the translation of an mRNA molecule that has already been transcribed. Mechanisms that use regulatory molecules that alter the length of time an mRNA survives before it's degraded by ribonucleases, affect translation initiation, or affect elongation factors and other proteins during the translation process. DNA ---> mRNA --x-- protein ---> activated protein.

transcriptional control

Allows variation in mRNA levels for specific enzymes. The cell avoids the production of these enzymes by utilizing regulatory proteins that prevent RNA polymerase from binding to a promoter. DNA --x-- mRNA ---> protein ---> activated protein.

indicator plates

Alternative strategy where mutants w/ metabolic deficiencies are observed directly. Monod adds a compiund that is acted on by beta-galactosidase. The compound acts as an indicator molecure for the presence of functioning beta-galactosidase because one of the molecules produced by the rxn is yellow. Colonies that stay white are unable to process the indicator molecule, meaning they have a defect in the beta-galactosidase enzyme or its production.

constitutive mutants

Cells that are abnormal because they produce a product at all times.

mechanisms of regular

DNA -- (transcription) --> mRNA -- (translation) --> protein -- (post-translational modifications) --> activated protein... slower response ---> rapid response

CAP binding site

DNA sequence that acts as a regulator switch; example of positive control. Located just upstream of the lac promoter.

What did Jacob & Monod's experiment find?

E. Coli colonies that grow on master plate (w/ glucose present) but are missing on the replica plate (w/ lactose only) represent mutants deficient in lactose metabolism.

What is the control of lactose metabolism?

E. coli can metabolize lactose (a disaccharide): it is transported into the cell and then cleaved to make monosaccharides (glucose and galactose). The enzyme beta galactosidase cleaves lactose, making glucose and galactose. Normally, beta galactosidase is not made unless lactose is present (and glucose is not).

adenylyl cyclase

Enzyme that produces cyclic AMP (cAMP) from ATP. Its' activity is inhibited by extracellular glucose.

Why is E. coli an excellent model organism for gene regulation?

It can grow on (metabolize) many different carbon sources. The enzymes for a particular carbon source are made (expressed) only when that carbon source is available.

replica plating

Key technique used in the search for mutants with defects in lactose metabolism. Begins with spreading mutagenized bacteria on a plate that's filled w/ gelatinous agar containing glucose (known as the master plate). Bacteria is allowed to grow, so that each cell produces a single colony. Next, a block covered w/ a piece of sterilized velve is pressed onto master plate. Due to contact, cells from each colony on the master plate are transferred to the velvet. Velvet is pressed onto a plate containing a medium that differes from that of the previous by a single component. Cells from the velvet stick to the plate's surface, producing an exact copy of the colonies on the master plate. This copy is the replica plate. After cells grow, investigator can compare the colonies that thrive on the replica plate's medium w/ those on the master plate.

What happens when a repressor is present and lactose present as well?

Lactose binds to repressor that was synthesized from normal lacI gene. The lactose-repressor complex causes it to release from DNA. Transcription occurs (w/ lactose acting as inducer by removing negative control). LacZ and Y can now be expressed in the mRNA.

polycistronic mRNA

Message that contains more than one protein-encoding segment.

inducer

Molecule that stimulates the expression of a specific gene or genes (ex: lactose acts as an enducer because it regulates the gene for beta-galactosidase, an enzyme that breaks sugars down into glucose and galactose).

lacY gene

Mutant phenotype: cells can't accumulate lactose inside the cell. Has defective copies of the membrane protein, galactoside permease, responsible for transporting lactose into the cell. This gene has much lower lactose concentration than normal cells. Under negative and positive control.

lacZ gene

Mutant phenotype: cells can't cleave indicator molecule even if lactose is present as an inducer. No beta-galactosidase activity. Gene for beta-galactosidase is defective. Under negative and positive control.

lacI gene

Mutant phenotype: indicator molecule is cleaved even if lactose if absent (no inducer). Prevents the transcription of lacZ and lacY when lactose is absent. Beta-galactosidase is made all the time (constitutive).

How does the CAP protein bind to the CAP building site and trigger transcription of the lac operon?

Not all promoters are created equal. Strong promoters allow efficient initiation of transcription by RNA polymerase; weak ones (like the lac promoter) support much less initiation efficiency. When the CAP regulatory protein is bound to CAP site just upstream of lac promoter, the protein interacts w/ RNA polymerase in a way that allows transcription to begin more frequently. CAP binding greatly strengthens the lac promoter. In this way, CAP exerts positive control of lac operon. When CAP is active, transcription increases.

induction (aka positive control)

Occurs when a regulatory protein beinds to DNA and triggers transcription.

post-translational control

Occurs when the cell controls the activity of an existing protein by chemical modification. Some proteins are manufactured in an inactive form and have to be activated by chemical change such as addition of a phosphate group, lipid group, carbohydrate, or folding. DNA ---> mRNA ---> protein --x-- activated protein.

Why is the lac Operon model important?

Regulation of the lac operon provided an important model system in genetics. We now know that gene expression of many bacterial operons is regulated by physical contact between regulatory proteins and specific regulatory sites on DNA. In addition, as in the lac operon, the activity of many other key regulatory proteins is regulated by post-translational control.

operator

Section of DNA in the lac operon where the repressor from lacI binds.

catabolite repression

Situation where transcription of the lac operon is drastically reduced when glucose is present in the environment-- even when lactose is available to induce beta galactosidase expression. Mechanism of glucose regulation.

repressor

Transcription inhibitor that was thought to bind directly to DNA near or on the promoter for the lac Z and Y gene (the lacI gene produces an inhibitor that exerts negative control over the lacZ and lacY gene)

What happens when trptophan is absent?

Transcription normally occurs and the trp operon expressed contains five genes coding for enzymes involved in tryptophan synthesis.

What happens when no repressor is present and lactose is either present or absent?

Transcription proceeds normally. Mutant lacI gene in the DNA do not produce a functional repressor. RNA polymerase bound to a promoter continues transcription and mRNA expressed lacZ and lacY.

What happens when trytophan is present?

Trytophan binds to repressor protein. The trytophan-repressor complex binds to the operator and shuts down transcription, ending the synthesis of trytophan and no more transcription of trp operon.

trp operon

Under negative control like the lac operon. Instead of coding for enzymes that perform catabolism (breakdown of lactose), they code for enzymes that perform anabolism (synthesis of trytophan).

gene expression

When a protein or other gene product is synthesized and is active in the cell.

negative control

When a regulatory protein binds to DNA and shuts down transcription.

allosteric regulation

When a small molecule binds directly to a protein and causes it to change its shape and activity.

In bacteria, how do low glucose levels affect adenylyl cyclase, cAMP, CAP, and transcription?

When extracellular concentration of glucose is low, there is an increase in the intracellular level of cAMP and an active adenylyl. The CAP-cAMP complex forms, binds to the CAP site, and allows RNA polymerase to intiate transcription efficiently. As long as the repressor isn't bound to the operator, transcription of lac operor is initiated frequently and lactose can be employed as an alternative energy source.

In bacteria, how do high glucose levels affect adenylyl cyclase, cAMP, CAP, and transcription?

When glucose is abundant outside the cell, adenylyl activity is low or inactive. Therefore, cAMP levels inside the cell are low. CAP is not in a CAP-cAMP complex, so it does not have the conformation that allows it to bind to the CAP site and stimulate lac operon. Infrequent transcription and the cell continues to use glucose as energy source. high glucose = low adenyl cyclase activity = low cAMP.

What are the three important types of lactose metabolism mutants (aka lac mutants)?

genotypes lacZ-, lacY-, and lacI-