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Select a topic from the following choices: Human stem cells Genetically modified foods Climate change Use of vaccinations Cloning Designer babies Consult resources other than the course textbook and this study guide. Examples of resources include other textbooks, journal articles, and reputable websites. Reputable online sources include encyclopedias and university or governmen tal websites. Never use Wikipedia as a source for a research paper. Prepare a detailed report on the topic you choose, including the following elements: 1. Describe the background and basic issue of your chosen topic 2. Describe the basic argument of opposing viewpoints. 3. Identify the strengths and weaknesses of both views. 4. Choose which view you agree with. (Only in the conclusion) 5. Explain why you agree with that view. Incorporate the information you learned about your chosen topic from this course, but don't limit your discussion to the textbook and study guide. Take advantage of external sures to providethcmprehensive report

Answer 1

Regulation and autoregulation by lexA protein

Author: Roger Brent

Introduction

The lexA gene’s product in E.coli inhibits many genes’ expression unless there’s DNA damage. LexA apart from repressing the himA, uvrA, uvrB, sfiA, and umuC genes also represses itself.

Lex A is expressed during SOS response (such as DNA damage). It is cleaved by the product of the recA gene which is transcribed in SOS response, now the lexA inhibited genes can be expressed.

This paper discusses three aspects of lexA protein’s action as a repressor: how it regulates other genes induced during the SOS response, how it regulates its own synthesis, and how it recognizes its operator sites.

lexA regulation of SOS functions

The repression mechanism by LexA protein is similar to what other repressors show. LexA protein binds to operator sites in front of the genes that have to be repressed, for two lexA-repressed genes so far examined, binding of LexA protein inhibits the binding of RNA polymerase to the promoter.

The different operator sites have different affinity for the LexA protein, the affinities are measured using a variant of the DNase protection technique. Affinity is known for operator sites in front of four lexA- repressed genes: recA, uvrB, sfiA, and the lexA gene itself. LexA protein binds most tightly to the operator site in front of the recA gene, and less tightly to the operator sites in front of uvrB, sfiA, and lexA.

The concentration of the lexA protein falls drastically after a major DNA damage. Intermediate levels of the protein are observed after mild DNA damage, at the beginning and at the end of an SOS response to drastic DNA damage, and after DNA damage in recA430 mutant cells deficient in recA protease activity. Because different operators have different affinities for lexA protein, it has been proposed that at intermediate LexA protein concentrations many lexA-repressed genes might be induced, but that the recA gene, whose operator binds lexA protein tightly, would not be induced.

The genes of SOS response are regulated in some lexA independent ways as well. For example, UvrB maintains a high un-induced level of expression with a second, lexA independent promoter. HimA, a gene whose product is required for the integration of bacteriophage lambda, seems to be repressed both by lexA protein and by a complex of its own gene product and another host protein (called hip or himD product).

Autoregulation of lexA synthesis

lexA transcription in vivo can be observed by using a bacteriophage lambda in which the lexA promoter is fused with the lacZ gene. lexA gene which is normally repressed and is expressed during DNA damage has two operator sites for LexA protein, in contrast with the single operator site found for the other six lexA-repressed genes. The protein binds to the two operator sites in front of lexA coordinately.

The two operator sites of the LexA binding protein were mutated and experiments were performed. It has been observed that the binding of the LexA protein to the two operator sites is cooperative, it requires both the sites to be functional in order to express the gene. If the operators are kept isolated or one is mutated and one is wild-type or if the cytosines in one of the operator are methylated, then the affinity of the operator is reduced and is less than that of the wild-type case.

With respect to the methylate of Cytosines in the operator sequence, it is possible that the basal level of lexA protein is lower in strains of E. coli that do not methylate cytosine, and that SOS functions might be easier to induce in these strains.

Physical aspects of protein binding

All eight lexA protein operator sites which have been sequenced possess approximate twofold rotational symmetry. The center of symmetry passes between two bases in the operator sites. Dimethyl sulfate protection experiments with LexA protein and five wild- type operators show that LexA protein protects the N7 position of two loose clusters of roughly symmetrically disposed of G’s in the operator sites. lexA protein protects no A’s, which means that the only functional groups lexA protein protect from dimethyl sulfate attack lie in the major groove of the DNA. The protected Guanine bases are bound 9 bases out from the center of symmetry so it can be concluded that the protein binds to a region that is 18 base pairs long.

When running on a sizing column, it is observed that LexA is a dimer with its carboxyl-terminal portion showing substantial homology to the carboxyl terminal of other bacteriophage repressors. The LexA protein’s N-terminal accounts for the repression activity only in sufficiently large quantities. LexA protein contacts DNA with groups in its amino terminus but requires an intact carboxyl terminus in order to dimerize and bind DNA strongly.

Three mutations in the operators in front of the lexA gene have unexpectedly small effects on repressor binding. The mutations are multiple substitutions which drastically alter the operator sequence. If the LexA control region is visualized so that the lexA gene itself is on the right, these mutations lie in the right half of either lexA operator site, while analogous mutations which abolish measurable repressor binding change the left half of either site. It is possible that the contacts that lexA protein makes with one half of an operator site are much more important than the contacts the protein makes with the other half.

There are two observations consistent with this idea: First if all sequenced LexA protein operators are aligned such that the genes they repress are transcribed from left to right, comparison of the operator sequences shows that the bases in the right half of an operator site are less likely to be conserved among all the sites. Second, the strongest Operator mutation known for the recA gene lies in the left half of the site.

Another line of evidence suggests that the LexA protein binds differently to the DNA than the other repressor proteins. The DNA binding proteins have a common feature, the super-secondary structure made up of two alpha helices held at a conserved angle with one of the two fittings into the major groove of the DNA.

Comparisons of the sequence of various phage repressor, cro, and other bacterial repressor proteins have shown that they all possess the double alpha helices but it is not the case with the LexA protein.

Even though the carboxyl-terminal has some homology, nothing in the lexA sequence predicts a helix which corresponds to the helix in the other proteins which fits into the major groove. It is possible that the amino terminus of lexA protein interacts with DNA by a different mechanism.

Fine control of the SOS response

LexA suppresses the SOS genes which are induced under certain circumstances. LexA protein binds weakly to operators of some genes like uvrB and sfiA and tightly to the recA operator.

In normal cells, lexA represses itself by weakly binding to the two operator sites, in a cooperative manner. Measurements of the amount the lexA promoter is repressed in vivo suggest that cooperativity makes the lexA promoter easier to de-repress than it otherwise would be. These facts have led to a picture of how the concentration of lexA protein can affect the response of a cell to DNA damage.

The expression of these SOS genes can be divided based on the magnitude of DNA damage sustained by the cell:

Minor DNA Damage

The DNA repair enzymes are sufficient to repair the DNA and the LexA level decreases very little. The rate of transcription of lexA is sufficient to again restore the LexA concentration.

Massive DNA Damage

Rate of lexA cleavage will exceed the rate of transcription of new LexA. LexA binds tightly to the operator of recA so the production of recA will take place only when the lexA is completely destroyed. Production of large amounts of recA protein may be necessary for efficient induction of lambda-like prophages and for efficient repair of DNA.

Intermediate DNA Damage

There is an intermediate concentration of LexA in the cell. If DNA damage is prolonged, then the cell will maintain some functional LexA protein only if the rate of LexA protein synthesis exceeds the rate of cleavage. Operators of some of the SOS genes bind weakly to LexA while some bind tightly, so genes like uvrB will be induced for DNA repair and recA gene will not be induced.

It is interesting to observe that the system used by the lexA protein to regulate its own concentration is correct, then cooperativity and autogenous regulation act to make it difficult to induce the SOS system maximally, while the same ways of controlling genes work to make some bacteriophages easy to induce.