Evidence for co-transcription of the RNA polymerase genes rpoBC with a ribosomal protein gene of escherichia coli.

The adjacent genes rpoB and rpoC code for the beta and beta' subunits of RNA polymerase in Escherichia coli, and are cotranscribed in the order given. The nearest known genes to rpoB are rplL and rplA,J,K, which code for ribosomal proteins, and which are transcribed in the same direction as the polymerase genes. It has been suggested that rpoBC may be distal elements of a larger operon including these ribosomal genes. To test this possibility we have cloned a segment of DNA, derived by endoR. HindIII digestion from the rpoBC-transducing bacteriophage lambdarifd18, in the replacement vector NMlambda761. The structure of the lambdarpoBC bacteriophages so produced is such that the inserted DNA can be transcribed from lambda promoters, allowing us to confirm that it carries intact rplL, rpoB, and rpoC genes. We have studied these bacteriophages as lysogens in rec+ and rec bacteria, and by infection of UV-irradiated bacterial strains in which lambda promoters are either repressed or active. The results indicate that the cloned DNA contains at most a very weak promoter for the above genes, in contrast to that present in the larger segment of bacterial DNA carried by lambdarifd18. We have in the same way cloned the adjacent bacterial HindIII-fragment of lambdarifd18 DNA, and have found that it displays vigorous autonomous expression of the tufB, rplA, and rplK genes. We conclude that rpoB and C are obligatorily co-transcribed with rplL, from a promoter located outside the DNA segment cloned in lambdarpoBC. We discuss the evidence for the existence of a regulatory site, rpoU, located between rplL and rpoB.     

Molecular analysis of a 21.1-kb fragment of wheat chloroplast DNA bearing RNA polymerase subunit (rpo) genes.

The entire nucleotide sequence of a 21.1-kb fragment of wheat chloroplast (ct) DNA was determined. This fragment carries 18 intact genes and parts of two additional genes, including the three RNA polymerase genes rpoB, rpoC1, and rpoC2. The gene arrangement of this region is conserved in wheat, rice, and maize, but not in non-grass species. Comparison of these 20 genes in wheat, rice, and maize showed that tRNA genes evolved more slowly than protein-coding genes in the chloroplast genome. Intergenic regions evolved much faster than both types of genes. Although the 19 genes of wheat, except for orf42, showed high identity to those of other plants, there were three novel structural features in the wheat rpoC2 gene; a deletion of 81 bp in the middle region, a variable insertion (408 bp), and a nonsense mutation in the 3' terminal region, resulting in truncation of a sequence of ca. 10 amino acids. An intermolecular recombination between the stretches of CTTAT and CTTTT was suggested as the mechanism of the 81-bp deletion in the wheat rpoC2 gene. Evolutionary distance between the chloroplast genomes of wheat and maize was larger than those between wheat and rice and between rice and maize.     

New transducing phage with RNA polymerase beta- and beta'-subunit genes derived from a hybrid phage lambda att80: isolation, genetic analysis and physical mapping]

A hybrid lambda att 80 phage with the genetic structure lambda (A-J) phi 80 (att-int-xis) imm lambda..cI857s7 is shown to be a convenient vector for creating transducing phages. On the one hand, the restriction analysis indicates that it has 3 restriction sites for EcoRI in comparison with 5 and 9 sites for parental phages lambda and phi 80 respectively. On the other hand, its buoyant density is less than that of phage lambda and under centrifugation it is easier separated from the phage transducing particles. When lambda att 80 prophage was excluded from the bfe locus of Escherichia coli, transducing phages with genes of two RNA polymerase beta-subunits (rpoB and rpoC) were isolated. To identify the latter, a convenient genetic test was worked out. A physical map of lambda att 80 drifd 35 transducing phage, carrying rpoB and rpoC genes has been constructed using endonucleases EcoRI and HindIII. A comparison of this map and the corresponding maps of transducing phages lambda drifd 18 and lambda drifd 47, studied earlier, led to the discovery of two integration sites of phage lambda within the locus bfe spaced apart by about 1800 nucleotide pairs. At all the sites both phages (lambda and lambda att 80) have integrated in the locus bfe in the counter clockwise order.