Question

Describe how you would identify genes responsible for transcription in E. coli by isolating mutations in their genes. Remember that these genes are required for cell viability. Include all the steps in the isolation of the mutants and also describe how you will differentiate the mutants you want from those in other genes necessary for viability. This includes giving me the expected result for mutants in the genes you want.

Answer 1

Answer:

E. coli have provided the subsequent investigations of transcription in eukaryotic cells. mRNA was discovered first in E. coli. E. coli was also the first organism from which RNA polymerase was purified. The basic mechanisms by which transcription is regulated were likewise elucidated by pioneering experiments in E. coli, in which regulated gene expression allows the cell to respond to variations in the environment, such as changes in the availability of nutrients. Transcription in E. coli provided more complex mechanisms that regulate gene expression in eukaryotic cells.

The DNA sequence to which RNA polymerase binds to initiate transcription of a gene is called the promoter. The DNA sequences involved in promoter function were first identified by comparisons of the nucleotide sequences of a series of different genes isolated from E. coli. These comparisons revealed that the region upstream of the transcription initiation site contains two sets of sequences that are similar in a variety of genes.

In the isolation of mutants that were defective in regulation of the genes involved in lactose utilization. These mutants were of two types: constitutive mutants, which expressed all three genes even when lactose was not available, and noninducible mutants, which failed to express the genes even in the presence of lactose. Genetic mapping localized these regulatory mutants to two distinct loci, called o and i, with o located immediately upstream of the structural gene for β-galactosidase. Mutations affecting o resulted in constitutive expression; mutants of i were either constitutive or noninducible.

The function of these regulatory genes, in which two strains of bacteria were mated, resulting in diploid cells containing genes derived from both parents. Analysis of gene expression in such diploid bacteria provided critical insights by defining which alleles of these regulatory genes are dominant and which recessive. For example, when bacteria containing a normal i gene (i+) were mated with bacteria carrying an i gene mutation resulting in constitutive expression (an i- mutation), the resulting diploid bacteria displayed normal inducibility; therefore, the normal i+ gene was dominant over the i- mutant. In contrast, matings between normal bacteria and bacteria with an oc mutation (constitutive expression) yielded diploids with the constitutive expression phenotype, indicating that oc is dominant over o+. In which mutations in o and i were combined with different mutations in the structural genes showed that o affects the expression of only the genes to which it is physically linked, whereas i affects the expression of genes on both chromosome copies in diploid bacteria. Thus, in an oc/o+ cell, only the structural genes that are linked to oc are constitutively expressed. In contrast, in an i+/i- cell, structural genes on both chromosomes are regulated normally. These results led to the conclusion that o represents a region of DNA that controls the transcription of adjacent genes, whereas the i gene encodes a regulatory factor (e.g., a protein) that can diffuse throughout the cell and control genes on both chromosomes.

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