Genetics Notes
Chapter 16 - Control of Gene Expression: Prokaryotes

These notes are provided to help direct your study from the textbook. They are not designed to explain all aspects of the material in great detail; that is what class time and the textbook is for. If you were to study only these notes, you would not learn enough genetics to do well in the course.

Gene expression and control
The control of gene expression primarily involves the control of transcription. mRNAs in bacteria are rapidly synthesized, broken down, and resynthesized. An E. coli mRNA is usually degraded within about 2 minutes of synthesis. This rapid turnover allows a very fine control over the quantity of any given mRNA and hence, the amount of any given protein product.

Not all proteins that a cell can make are needed all the time. For example, it would be wasteful of the cell to produce the enzymes necessary to produce histidine if histidine was present in the media.

There are two basic control systems.
1) repressible system - transcription of the proteins needed for a synthetic (anabolic) pathway are blocked when the end product is in sufficient supply (figure 16.5).
2) inducible system - transcription is turned on when the starting product for a degradation (catabolic) pathway is present (figure 16.4).
Inducible system
Lactose (milk sugar) is a beta-galactoside (disaccharide) and it can be used for energy by E (figure 16.6). coli. The enzyme responsible for the breakdown is beta-galactosidase. Its gene is called lacZ. Two other proteins are that turned on when this system is induced are:
beta-galactoside permease (lacY), which is a transport protein to bring lactose into the cell.
beta-galactoside acetyl transferase (lacA), which protects cells from toxic products
These 3 enzymes are arranged sequentially on the E. coli chromosome and are in the same transcript (piece of RNA) (figure 16.7).

In the DNA of E. coli, there is a fourth gene, the regulator gene, lacI. The regulator gene is under separate transcriptional control from the other three genes. lacI codes for a protein called a repressor. The repressor binds to a region just before the lacZ gene. This region is called the operator.

This sequence of bases in the DNA is recognized by the repressor. The promoter is the same basic sequence of nucleotides for initiation of transcription that we studied in Chapter 13 (figure 16.8). Thus, the specific definition of an operon is a sequence of genes all under the transcriptional control of the same operator (figure 16.3).

The repressor blocks binding of RNA polymerase and thus blocks transcription (figure 16.7). When lactose is present, some of it is converted to allolactose. Allolactose then binds to the repressor protein. This changes the shape of the repressor and it disassociates from the operator region. This frees RNA polymerase to begin transcribing the lac operon. Proteins that change shape when a molecule binds to it are called allosteric proteins.

Catabolite Repression
Another interesting point about the lac operon is that it is turned off if glucose is present even if lactose is also present. When glucose is at low concentrations in the cell, cyclic AMP concentrations are high (figure 16.12).

Cyclic AMP binds with a protein called catabolite activator protein (CAP). When cAMP binds with CAP, this complex then binds with part of the promoter region (figure 16.12). This binding facilitates binding of RNA polymerase and thus increases the rate of transcription. When glucose is high, cAMP is low and the cAMP-CAP complex is not available to bind to the promoter. This allows the cell to use glucose as the preferred substrate and lactose is used as the glucose runs out (figure 16.12).

Mutants of the lac operon
In constitutive mutants, the lac operon genes are continuously transcribed. A mutant regulator gene (lacI-) produces a defective repressor that cannot bind to the operator. A mutant operator region (lacOc) is defective in that the repressor cannot bind and thus transcription cannot be shut off. Both types of mutants have the same constitutive phenotype.

It is possible to determine whether a constitutive mutant is a regulator or an operator mutant by the production of merozygotes. Some strains of E. coli have the lac operon incorporated into a F' factor. The F' strains can pass the F' factor plus the lac operon into other F- strains. This can produce cells that are functionally diploid for the lac operon (figures 16.9 and 16.11).

If a cell has lacI- lacO+ lacZ+ on its chromosome and a F factor carrying lacI+ lacO+ lacZ+ is transferred to the cell. Then a merozygote (merodiploid) results that is lacI- lacO+ lacZ+/lacI+ lacO+ lacZ+. This cell has a mutant lacI gene. If the piece of DNA with lacI- was present in a cell by itself, it would be constitutive. Note that the protein product produced by the lacI+ gene on the F' factor produces sufficient protein product to control transcription of both operons. Therefore, lacI+ is dominant to lacI-. Because lacI+ is dominant to lacI-, constitutive regulator mutants are said to be trans-dominant or tans-acting (figure 16.9).

If the chromosome has a mutant operator region, the cell continuously transcribes the lac operon because the repressor produced by the lacI+gene of both pieces of DNA cannot bind to the operator region on the chromosome (lacI+ lacOc lacZ+/lacI+ lacO+ lacZ+). lacOc is dominant to lacO+ and because of this, the constitutive operator mutants are called cis-dominant or cis-acting (figure 16.9).

In addition to these mutants, there are
lacIs is a super-repressed mutant in which the repressor does not bind lactose and thus will not disassociate from the operator region (figure 16.10). lacIQ produces much more repressor than is normal, and is presumably a mutation in the promoter region of the i gene.
Repressible system
Control of repressible systems is accomplished through two separate control mechanisms.
1) control of transcription via repressors similar to that for lac operon
2) control or attenuation of translation by an attenuator region in the mRNA of the operon
Operator control
In the tryptophan operon, there are 5 genes that code for the 3 enzymes needed to convert chorismic acid to tryptophan (figure 16.14). In addition, the trpR gene produces a repressor protein which binds to tryptophan. After binding tryptophan the repressor can bind to the operator region. This then blocks RNA polymerase from transcribing the trp operon. As tryptophan decreases in concentration, the tryptophan begins to disassociate from the repressor. The repressor can no longer bind to the operator and it disassociates. In this system, the end product (tryptophan) represses transcription.

Attenuator control
Yanofsky noticed that in constitutive mutants of the trpR gene, tryptophan also blocked transcription by some mechanism. As it turns out, the attenuator region is about 90 base pairs long and contains four very distinct regions that can cause loop structures within the mRNA (figures 16.15 and 16.16). This piece of mRNA is also translated under certain condition into a small peptide of 14 amino acids. This region is called the leader peptide gene (figure 16.16)

If the operator is unblocked, translation begins. As soon as the 5' end of the leader peptide has been transcribed, a ribosome attaches and translation begins.

There are 3 possible outcomes.
1) If the concentration of tryptophan is high, such that tryptophanyl-tRNA is present, translation will proceed down the leader peptide. The ribosome will overlap regions one and two and this allows strong binding between 3 and 4. The stem loop structure between 3 and 4 will cause transcription to terminate (figure 16.16). Of course, this only happens if the repressor gene or the operator is non-functional because excess tryptophan should block transcription at the operator.

2) If there is a reduced quantity of tryptophanyl-tRNA, the ribosome will stall at position 9 or 10 in the translation of the leader peptide. Positions 9 and 10 are both tryptophan. This allows region 2 to pair with region 3. This opens up the loop that would form if 3 and 4 were paired. Thus, if tryptophan is limited, the RNA polymerase can bypass the terminator loop at 3 and 4 and the operon will be transcribed (figure 16.16).

3) general starvation: In this situation, no amino acids are available. Thus, it does not make any sense to transcribe the tryptophan operon to synthesize tryptophan if the other amino acids are also in short supply. In this case, the ribosome will never start translation and it will never get into the first region. Since pairing between 1 and 2 is not disrupted, 3 and 4 are also paired. Therefore, the terminator loop is functional and transcription is blocked. This mechanism allows the cell to carefully match the quantities of the amino acids to each other.
Additional control mechanisms

promoter efficiency
The promoter sequence of different genes in E. coli are different. The affinity of RNA polymerase is different for different promoter sequences. The higher the affinity, the higher the rate of transcription. For example, the lacI gene of the lac operon is a constitutive gene in that it is not under control by any operator region. However, its promoter region has a low affinity for RNA polymerase and only 1 mRNA is made per cell cycle. The lacIQ mutant has a more efficient promoter region and can get up to 50 mRNA's per cell cycle. Enzymes needed in large quantities are coded by genes that have more efficient promoter regions.

translational control
There is little control of protein synthesis after the mRNA is made. However, it has been noted that for the lac operon, the 3 genes are translated in the ratio of 10:5:2. The first gene is translated at a higher rate than the third gene. Apparently, this is due to the fact that translation can occur before transcription has been completed. Genes transcribed first have more time to be translated as compared to the last genes in the operon. Natural selection may have selected for the optimal order of genes in the operon such that those needed in the greatest quantities are transcribed first.

Last update on 26 November 2004
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