Clustering of tRNA cistrons in Escherichia coli DNA

Characterization of tRNA:DNA hybrids reveals that many, perhaps most, of the tRNA genes in $\text{E. coli}$ DNA are clustered. Density and double-isotope measurements show that 3–4 molecules of tRNA can hybridize with DNA fragments that are only 4–5 times larger than a mature tRNA. Treatment of the hybrids with a single-strand-specific endonuclease results in the solubilization of 30–35% of the DNA and the formation of monocistronic hybrids.     

Separation of E. coli tRNA-Tyr precursor RNA from E. coli tRNA by hydroxyapatite chromatography

An assay procedure is described for triosephosphate isomerase based on measurement of the ellipticity of l-glyceraldehyde 3-phosphate remaining when d,l-glyceraldehyde 3-phosphate is the source of substrate and d-glyceraldehyde 3-phosphate is converted by triosephosphate isomerase to dihydroxyacetone phosphate. The assay method has advantages over the conventional coupled-enzyme assays in that it circumvents the difficulties posed by instability of the coupling enzymes and their cofactors, as well as by inhibitors of triosephosphate isomerase which may be present in preparations of the coupling enzymes. Although the method is not suited for routine assays during purification or in most clinical applications, it has advantages for detailed kinetic studies where pH, temperature, or other factors cause the coupled-enzyme assay procedures to be unreliable.     

Misacylation of yeast amber suppressor tRNA(Tyr) by E. coli lysyl-tRNA synthetase and its effective repression by genetic engineering of the tRNA sequence

Through an exhaustive search for Escherichia coli aminoacyl-tRNA synthetase(s) responsible for the misacylation of yeast suppressor tRNA(Tyr), E. coli lysyl-tRNA synthetase was found to have a weak activity to aminoacylate yeast amber suppressor tRNA(Tyr) (CUA) with L-lysine. Since our protein-synthesizing system for site-specific incorporation of unnatural amino acids into proteins is based on the use of yeast suppressor tRNA(Tyr)/tyrosyl-tRNA synthetase (TyrRS) pair as the "carrier" of unusual amino acid in E. coli translation system, this misacylation must be repressed as low as possible. We have succeeded in effectively repressing the misacylation by changing several nucleotides in this tRNA by genetic engineering. This "optimized" tRNA together with our mutant TyrRS should serve as an efficient and faithful tool for site-specific incorporation of unnatural amino acids into proteins in a protein-synthesizing system in vitro or in vivo.     

Double mutations induced in Escherichia coli by ultraviolet light.

Double mutations to azide resistance and to bacteriophage T5 resistance of genes separated by more than 50 kilobases were induced in Escherichia coli WP2s in chemostat cultures by exposure to a single low dose of ultraviolet light. Frequencies of induced double mutations were three orders of magnitude greater than would be predicted by chance. Reversions from azide resistance and phage resistance occurred independently, showing that that the double mutation was not due to pleiotropic effects of a single gene mutation. These results support earlier findings which show that low doses of ultraviolet light induce multiple gene mutations in Bacillus subtilis over a similarly broad range.     

Recognition of E coli tRNAArg by arginyl tRNA synthetase.

Escherichia coli tRNAArg was digested with ribonuclease T1 under restrictive conditions in order to dissect a minimum number of diester bonds. The number of diester bonds cleaved and their locations were determined by phosphorylation of the newly formed 5' hydroxyl groups with [32P] ATP and polynucleotide kinase. There was complete loss of aminoacylation of tRNAARg when two diester bonds were cleaved at the anticodon. However, this material retained the specific properties of synthetase recognition. Two fragments were derived by further digestion of this tRNA. One 19 nucleotide-long fragment derived from the 3' end of tRNAArg and another 18 nucleotide-long fragment derived from the 5' end of the molecule were required to maintain the properties of the specific recognition by the arginyl tRNA synthetase in the absence of the rest of the structure including the anticodon.     

Conformational changes of yeast tRNAPhe and E. coli tRNA2Glu as indicated by different nuclease digestion patterns.

The susceptibility of yeast tRNAPhe and Escherichia coli tRNA2Glu to digestion by nucleases Tl and Sl are examined in a variety of environments, and the results are interpreted in view of the available three-dimensional structural information. Significant differences are found in the digestion pattern of the two tRNAs using the guanosine-specific Tl nuclease. In particular, differences are seen due to varying the type of salts in the environment. However, the Sl nuclease results on the two tRNAs do not differ greatly. E. coli tRNA2Glu is known to exist in two different conformations. Nuclease digestion results are presented revealing differences which make it possible to draw some inferences about the structural differences in these two conformations. In carrying out these analyses, the tRNA molecules are labeled either by putting 32P at the 5'-end of the molecular or by adding 32P-labeled pCp at the 3'-end. It is found that both yeast tRNAPhe and E. coli tRNA2Glu have modified Tl nuclease digestion patterns when pCp is added at the 3'-end of the molecule.     

Modification of specific lysine residues in E. coli methionyl-tRNA synthetase by crosslinking to E. coli formylmethionine tRNA

A protein affinity labeling derivative of E. coli tRNAfMet has been prepared which carries an average of one reactive side chain per molecule, distributed over four structural regions. Each side chain contains a disulfide bond capable of reaction with cysteine residues and an N-hydroxysuccinimide ester group capable of coupling to lysine epsilon-amino groups in proteins. Reaction of the modified tRNA with E. coli methionyl-tRNA synthetase leads to crosslinking only by reaction with lysine residues in the protein. Examination of the tRNA present in the crosslinked complex reveals that the enzyme is coupled to side chains attached to the 5' terminal nucleotide, the dihydrouridine loop, the anticodon and the CCA sequence. Digestion of the crosslinked enzyme with trypsin followed by peptide mapping reveals that the major crosslinking reactions occur at four specific lysine residues, with minor reaction at two additional sites. Native methionyl-tRNA synthetase contains 90 lysine residues, 45 in unique sequences of the dimeric alpha 2 enzyme. Crosslinking of the protein to different regions in tRNAfMet thus occurs with the high degree of selectivity necessary for use in determining the peptide sequences which are near specific nucleotide sequences of tRNA bound to the protein.     

Nucleotide sequence and transcriptional organization of the Escherichia coli enterobactin biosynthesis cistrons entB and entA

The nucleotide sequence of a 2,137-base-pair DNA fragment expressing enterobactin biosynthesis functions defined the molecular boundaries and translational products of the entB and entA genes and identified a closely linked downstream open reading frame encoding an uncharacterized protein of approximately 15,000 daltons (P15). The sequence revealed that an independent protein-coding sequence corresponding to an EntG polypeptide was not situated in the genetic region between the entB and entA cistrons, to which the EntG- phonotype had been genetically localized. As a result, the biochemical nature of the EntG function in the biosynthetic pathway requires reevaluation. The EntA polypeptide displayed significant similarities at the amino acid level to the pyridine nucleotide-binding domains of several members of a family of alcohol-polyol-sugar dehydrogenase enzymes, consistent with its function as the enzyme catalyzing the final step of dihydroxybenzoate biosynthesis. An additional role for EntA in the isochorismate synthetase activity of EntC was strongly implicated by genetic evidence. Evidence from the nucleotide sequence of this region and newly constructed ent-lacZ fusion plasmids argues strongly that these genes are linked in an iron-regulated entCEBA (P15) polycistronic operon.