A pair of Bacillus subtilis ribosomal protein genes mapping outside the principal ribosomal protein cluster.

Before now, the only ribosomal protein gene loci to be identified in Bacillus subtilis map within the principal ribosomal protein gene cluster at about 10 degrees on the linkage map. Using mutants with alterations in large subunit ribosomal proteins L20 or L24, I mapped the corresponding genes near leuA at approximately 240 degrees. The data were fully consistent with the fact that the genes for the two proteins were close together but not near any other ribosomal protein genes, as is also the case with the genes for the corresponding proteins of Escherichia coli.     

Zinc is associated with the beta subunit of DNA-dependent RNA polymerase of Bacillus subtilis.

The Bacillus subtilis DNA-dependent RNA polymerase holoenzyme and core enzyme each contain approximately two atoms of zinc per molecule. When the dissociated subunits of the enzyme are passed through a blue dextran-Sepharose affinity column, only the beta subunit binds to the column. The total zinc content of the enzyme is tightly bound to the beta subunit. Dialysis studies suggest that the two zinc ions differ in the strength of their association with the beta subunit. The presence of zinc in beta is consistent with several other lines of evidence which indicate that this subunit is dirrectly involved in phosphodiester bond formation. The blue dextran-Sepharose column procedure should be useful in future studies of the dissociation and reassociation of the enzyme since the method is rapid and provides excellent recovery of the beta subunit as well as the alpha and beta' subunits of the RNA polymerase.     

A cluster of nine tRNA genes between ribosomal gene operons in Bacillus subtilis.

A cluster of nine tRNA genes located in the 1-kb region between ribosomal operons rrnJ and rrnW in Bacillus subtilis has been cloned and sequenced. This cluster contains the genes for tRNA(UACVal), tRNA(UGUThr), tRNA(UUULys), tRNA(UAGLeu). tRNA(GCCGly), tRNA(UAALeu), tRNA(ACGArg), tRNA(UGGPro), and tRNA(UGCAla). The newly discovered tRNA gene cluster combines features of the 3'-end of trnI, a cluster of 6 tRNA genes between ribosomal operons rrnI and rrnH, and of the 5'-end of trnB, a cluster of 21 tRNA genes found immediately 3' to rrnB. Neither the tRNA(UAGLeu) gene nor its product has been found previously in B. subtilis. With the discovery of this new set of tRNA genes, a total of 60 such genes have now been found in B. subtilis. These known genes account for almost all of the tRNA hybridizing restriction fragments of the B. subtilis genome. The 60 known tRNA genes of B. subtilis code for only 28 different anticodons, compared with a total of 41 different anticodons for 78 tRNA genes in Escherichia coli. This may indicate that B. subtilis does not need as many anticodons because of more flexible translation rules, similar to the situation in Mycoplasma capricolum.     

Peptide mapping of Bacillus subtilis RNA polymerase alpha factors and core-associated polypeptides

An analysis of the peptide maps of the sigma factors and core-associated subunits of Bacillus subtilis RNA polymerase has revealed that all the sigma factors ad core-associated polypeptides are derived from separate genes and are not proteolytically modified products of the major 55,000-dalton sigma factor. A comparison of the peptide pattern of the major B. subtilis and Escherichia coli sigma factors revealed limited homology between them. Furthermore, antibody prepared against the 55,000-dalton B. subtilis sigma factor cross-reacted against the E. coli sigma factor, but not against any of the other B. subtilis sigma factors and core-associated polypeptides. These results unambiguously demonstrate the independently derived nature of the B. subtilis RNA polymerase core-associated subunits and the partial relationship between the major sigma factors of B. subtilis and E. coli.     

Chloramphenicol resistance mutation in Escherichia coli which maps in the major ribosomal protein gene cluster.

Localized mutagenesis and selection for streptomycin resistance were utilized to isolate a chloramphenicol resistance mutation in Escherichia coli K-12 linked to the strA (rpsL) locus. Bacteriophage P1 transduction verified the map position of the new resistance mutation at 72 min, placing it within a dense cluster of ribosomal protein genes. The map position differs from that of known cmlA and cmlB mutations, which map at 18 and 21 min, respectively. Ribosomes prepared from chloramphenicol-resistant and -sensitive isogenic transductants were analyzed in vitro for activity in formation of N-formylmethionyl-puromycin, polyphenylalanine, and polylysine in the presence of inhibitory concentrations of chloramphenicol. Comparisons were also made of 14C-chloramphenicol binding to 70S ribosomes and of the two-dimensional polyacrylamide gel electrophoresis pattern of ribosomal proteins from each strain. There was no detectable difference between ribosomes from sensitive and resistant strains as measured by these assays. Enzymatic modification by chloramphenicol acetyltransferase is not responsible for the observed phenotype.     

Precursors of 5 S ribosomal RNA in Bacillus subtilis

Bacillus subtilis 168 accumulates subnormal quantities of mature 5 S ribo-somal RNA in the presence of inhibitors of protein synthesis, such as chloramphenicol, or during pulse-labeling experiments. However, two RNA species, evidently precursors of m5 rRNA and therefore designated as p5_A and p5_B, do accumulate under these conditions. These RNA species are substantially longer than B. subtilis m5 rRNA: p5_A is about 179 nucleotides in length and p5_B is composed of approximately 152 nucleotides. The sum of p5_A, p5_B and m5 rRNA accumulating in the absence of protein synthesis, less excess chain length associated with p5_A and p5_B, equals the expected quantities of m5 rRNA in growing cells. p5_A and p5P_B both contain all t₁ RNase-generated oligonucleotides characteristic of m5 rRNA plus additional sequences. At least the 5′ termini of p5_A and p5_B differ from that of m5. If chloramphenicol is removed from a culture in which p5_A and p5_B have accumulated and further RNA synthesis is inhibited, then a quantitative reciprocal loss of p5_A and p5_B occurs as m5 rRNA accumulates. No evidence suggests any p5_A to p5_B transition under these conditions.     

The effect of the delta subunit on the interaction of Bacillus subtilis RNA polymerase with bases in a SP82 early gene promoter.

We have examined the effect of the delta subunit on the interaction of the Bacillus subtilis RNA polymerase with an early gene promotor of phase SP82. Methylation by dimethyl sulfate, used to probe close approaches of polymerase to purines, revealed that noninitiated complexes formed by holo-enzyme (core-sigma-delta) had significantly fewer contacts than complexes formed by core-sigma. The presence or absence of delta had little or no effect on close approaches to purines in initiated complexes. DNAase I footprinting indicated that core-sigma was bound to the same region regardless of whether delta, initiating nucleotides, or both, were present. These data support the conclusion that delta acts prior to initiation to enhance promoter selectivity by limiting the number of possible interactions that the polymerase can make with DNA.