Another type of mutant in the lac operson are the i- mutants, which have repressors which cannot bind the operator. Their phenotype is constant expression of the Z and Y genes. The is mutants have repressors which cannot bind lactose. Their phenotype is constant binding of the repressor to the operator, and a lack of expression in the Lac operon. When these mutants are brought together, the i gene mutants work in trans, while the O and P mutants only work in cis. Mutants acting only in cis are generally in sites on the DNA, while those which work in trans are generally coding for proteins.
An additional level of control of the lac operon can sense glucose levels. When glucose levels are high, it would be wasteful for the E. coli to transcribe the lac genes even if lactose were present, because glucose is a more efficient energy source. When glucose levels are low, the level of the small molecular cAMP (cyclic AMP) is high. This molecule can bind to a protein called CRP (cAMP receptor protein). The cAMP-CRP complex can bind near the promoter of the lac operon and facilitate transcription. This is an example of positive regulation of transcription.
Analysis of DNA sequences in the lac operon confirms the model developed based on genetic analysis (see Fig. 11.6 in text). Protection assays in which regions resistant to nuclease digestion as a result of protein binding have been used to define specific regions bound by the cAMP-CRP complex, by RNA polymerase and by the lac repressor.
The trp operon of E. coli illustrates control of gene expression in a prokaryotic biosynthetic pathway. This system was historically important because the trpA gene was studied and mutations in the gene were in the same order as amino acid substitutions in the protein. When the amino acid trp (tryptophan) is present, it binds to an aporepressor, which then binds to the trp operator, shutting down transcription. The system is repressible (the small molecule shuts off expression) and under negative control (binding of the protein shuts off transcription).
Phage lambda can follow either the lytic or lysogenic pathway. Mutants with clear plaques (instead of turbid plaques) were isolated which were defective in lysogeny. Some types of clear plaque mutants included operator mutants unable to bind a repressor; cI- mutatnts in which the repressor could not bind the operator; and mutations in genes related to lysogeny (cII- and cIII-). The actual process in lambda infection involves a balance between expression of genes favoring lysis (exemplified by cro) and expression of genes favoring lysogeny (exemplified by cI).
Eukaryotes differ from prokaryotes in several ways; these affect their gene expression patterns. (1) No polycistronic messages or operons. (2) Most of DNA is covered with histones. (3) Much repetitive DNA, most does not code for proteins. (4) DNA rearrangement or modification is sometimes used to control gene expression. (5) Introns are present. (6) Transcription and translation are uncoupled.
DNA alteration/ modification is sometimes used to regulate eukaryotic gene expression. Some examples follow.
Gene amplification of ribosomal DNA sequences can take place in animals with large eggs. This allows more rRNA to be produced than could be produced by the chromosomal genes alone. In Xenopus laevis, for example, the normal complement of 600 genes is increased to 2 million copies.
Yeast mating type switching is another example of DNA alteration for expression control. The a and alpha are the two mating types which can mate with each other. Yeast can sometimes switch types through a recombination mechanism in which a "cassette" of the opposite mating type gene is inserted into the active expression site.
Antibody genes are rearranged at the genomic level to allow a high diversity of antibody types to be produced. This can lead a few hundred germ line genes to be shuffled into ten to a hundred million different types of antibodies.