Recognition of messenger RNA during translational initiation in Escherichia coli

The structural aspects of recognition by E. coli ribosomes of translational initiation regions on homologous messenger RNAs have been reviewed. Also discussed is the location of initiation region on mRNA, its confines, typical nucleotide sequences responsible for initiation signal, and the influence of RNA macrostructure on protein synthesis initiation. Most of the published DNA nucleotide sequences surrounding the start of various E. coli genes and those of its phages have been collected.     

Expression of the lac operon in RNA polymerase mutants of Escherichia coli K12.

Summary Radioactively labeled 4.5S, 6S, and 10S RNAs from Escherichia coli were hybridized to EcoRI fragments from the E. coli genome. Each of these molecules bound to more than one DNA fragment. C_ot curve analysis of the kinetics of the annealing of these RNAs to denatured E. coli DNA suggests that the DNA corresponding to each of these molecules is reiterated in the genome. These experiments also suggest that these reiterated sequences are non adjacent.     

The relationship between translational initiation and messenger RNA inactivation in down-shifted Escherichia coli

The parameters of protein synthesis and functional inactivation of global messenger RNA (mRNA) were examined in a Tic+ strain of Escherichia coli during the 30-min period following a shift-down from glucose-minimal to succinate-minimal medium. The rate of mRNA inactivation and the relative translational initiation frequency were both most severely depressed immediately after the shift-down and increased slowly thereafter. If glucose was restored to the medium at any time after shift-down, mRNA inactivation immediately resumed its normal (preshift) rate and the protein-forming capacity was increased. These changes in mRNA inactivation rate do not reflect an altered mRNA composition in the down-shifted cells. The relative rate of mRNA inactivation was linearly proportional to the relative translational initiation frequency over a 10-fold range of initiation frequencies. Low initiation frequencies represent increased "dwell" of the ribosomes at the initiation site before the commencement of polypeptide chain initiation. We propose that initiating ribosomes protect mRNA from an inactivating endonucleolytic cleavage at or near the ribosome binding site.     

Lac messenger RNA in lac Z gene mutants of Escherichia coli caused by insertion of bacteriophage Mu

The messenger RNA made under conditions of induction of the lac operon has been studied in various lac mutants in which the mutation was caused by integration of bacteriophage Mu into the Z gene. The percentage of RNA hybridizing specifically to lac DNA is proportional to the distance from the beginning of the gene at which a given mutation is located. It thus appears that only lac RNA proximal to the site of insertion is transcribed in these mutants. This may account for the complete polarity of Mu-induced lacZ mutants.     

Construction of lac fusions to the inducible arginine-and lysine decarboxylase genes of Escherichia coli K12

The induction of several amino acid decarboxylases under anaerobic conditions at low pH has been known for many years, but the mechanism associated with this type of regulation has not been elucidated. To study the regulation of the biodegradative arginine and lysine decarboxylases of Escherichia coli K12, Mudlac fusions to these genes were isolated. Mudlac fusion strains deficient for lysine decarboxylase or arginine decarboxylase were identified using decarboxylase indicator media and analysed for their regulation of beta-galactosidase expression. The position of the Mudlac fusion in lysine decarboxylase-deficient strains has been mapped to the cadA gene at 93.7 minutes, while the Mudlac fusions exhibiting a deficiency in the inducible arginine decarboxylase have been mapped to 93.4 minutes.     

Models for decay of Escherichia coli lac messenger RNA and evidence for inactivating cleavages between its messages.

The size distributions of decaying polycistronic Escherichia coli lac messenger RNA have been followed on polyacrylamide gels. At the same time, equations have been derived that generate the theoretical size distributions of decaying macromolecules for different mechanisms of degradation. Using observed values of lac mRNA metabolism, it was possible to reproduce the in vivo patterns with a model in which cleavage occurs at the start of each of the three messages² and is followed by a net 5′ to 3′ wave of mass loss. Other models of degradation could not generate the observed in vivo patterns. These alternative mechanisms include: (1) the same number of primary cleavage sites (three) but at different positions on the full-length molecule; (2) an exclusive 5′ to 3′ directional degradation from the start of the lac mRNA (no cleavages); or (3) the presence of many internal targets. Further support for primary cleavage at the start of messages came from the observed accumulation of the intact z mRNA released by cleavage at the $\text{z}\text{y}$ boundary and from the predictable effects of specific deletions on the resultant size distributions.The significance of these cleavages has been assessed; they could be necessary “processing” events or, conversely, inactivate the message for translation. Full-length molecules as well as cleavage fragments have been fractionated by successive sucrose gradient centrifugation and tested for their capacity to form translation initiation complexes in vitro. Full-length lac RNA could form such complexes at one or more of its three ribosome-loading sites, whereas the y or a message fragments were inactive. These results suggest that a cleavage at or near the start of a message inactivates it.     

Catabolite repression in the D-serine deaminase system of Escherichia coli K-12

The induced synthesis of d-serine deaminase in Escherichia coli is subject to three catabolic effects: inhibition on inducer uptake, transient repression, and catabolite repression. Inhibition on d-serine uptake is not significant at the d-serine concentration normally used for induction. Transient repression and catabolite repression of d-serine deaminase synthesis are abolished by mutations in dsdCy, which appears to be an operator locus. The decline in the rate of constitutive synthesis observed in dsdCx mutants growing with glycerol as carbon source at temperatures above 37 C is due to catabolite repression. The low level of constitutivity at 37 C and the partial cis dominance of dsdCx mutants are not artifacts of catabolite repression. It is suggested that a product of one of the genes of the dsd operon may regulate the expression of the operon.     

lac Thiogalactoside transacetylase of Escherichia coli K-12 and ML

The lac thiogalactoside transacetylase was purified from both a wild-type Escherichia coli K-12 strain (H3000) and an E. coli ML strain (ML308). These enzymes are indistinguishable by using several criteria. The subunit molecular weight of the enzyme is 24,800, which is significantly less than the previously reported value of 30,000. Although the function of the thiogalactoside transacetylase is unknown, it is suggested that this enzyme plays an important role in lactose utilization since its structure and enzymatic activity have been conserved.