An internal region of rpoB is required for autogenous translational regulation of the beta subunit of Escherichia coli RNA polymerase.

In order to delineate the region involved in feedback regulation of the RNA polymerase beta subunit (encoded by rpoB), a collection of rpoB-lacZ translational fusions with different endpoints both upstream and downstream of the rpoB start site was assembled on lambda phage vectors. The extent of translational repression of beta was monitored by measuring beta-galactosidase levels in monolysogens of the fusions under conditions of increased intracellular concentrations of beta and beta' achieved via the induction of rpoBC expression from a multicopy plasmid. A construct containing as little as 29 bp upstream of the start of rpoB exhibited repression of beta-galactosidase activity to the same extent as a construct encoding the full upstream region. A construct which carried only 70 bp of the rpoB structural gene exhibited very little repression, while constructs which carried 126 or 221 bp of rpoB exhibited approximately the same degree of repression as a construct which carried 403 bp. These data suggest that the sequences important for feedback regulation of beta translation extend more than 70 bp into rpoB but are completely contained within a region which spans the sequences from 29 bp upstream to 126 bp downstream of the translational start site.     

Spinach chloroplast rpoBC genes encode three subunits of the chloroplast RNA polymerase

Sequence analysis of a 12,400 base-pair region of the spinach chloroplast genome indicates the presence of three genes encoding subunits of the chloroplast RNA polymerase. These genes are analogous to the rpoBC operon of Escherichia coli, with some significant differences. The first gene, termed rpoB, encodes a 121,000 Mr homologue of the bacterial beta subunit. The second and third genes, termed rpoC1 and rpoC2, encode 78,000 and 154,000 Mr proteins homologous to the N and C-terminal portions, respectively, of the bacterial beta' subunit. RNA mapping analysis indicates that the three genes are cotranscribed, and that a single intron occurs in the rpoC1 gene. No splicing occurs within the rpoC2 gene or between rpoC1 and rpoC2. Furthermore, the data indicate the possibility of an alternative splice acceptor site for the rpoC1 intron that would give rise to a 71,000 Mr gene product. Thus, with the inclusion of the alpha subunit encoded by rpoA at a separate locus, the chloroplast genome is predicted to encode four subunits (respectively called alpha, beta, beta', beta") equivalent to the three subunits of the core enzyme of the E. coli RNA polymerase.     

Evolutionary relationships among eubacteria, cyanobacteria, and chloroplasts: evidence from the rpoC1 gene of Anabaena sp. strain PCC 7120.

RNA polymerases of cyanobacteria contain a novel core subunit, gamma, which is absent from the RNA polymerases of other eubacteria. The genes encoding the three largest subunits of RNA polymerase, including gamma, have been isolated from the cyanobacterium Anabaena sp. strain PCC 7120. The genes are linked in the order rpoB, rpoC1, rpoC2 and encode the beta, gamma, and beta' subunits, respectively. These genes are analogous to the rpoBC operon of Escherichia coli, but the functions of rpoC have been split in Anabaena between two genes, rpoC1 and rpoC2. The DNA sequence of the rpoC1 gene was determined and shows that the gamma subunit corresponds to the amino-terminal half of the E. coli beta' subunit. The gamma protein contains several conserved domains found in the largest subunits of all bacterial and eukaryotic RNA polymerases, including a potential zinc finger motif. The spliced rpoC1 gene from spinach chloroplast DNA was expressed in E. coli and shown to encode a protein immunologically related to Anabaena gamma. The similarities in the RNA polymerase gene products and gene organizations between cyanobacteria and chloroplasts support the cyanobacterial origin of chloroplasts and a divergent evolutionary pathway among eubacteria.     

Mutation slowing down the degradation of the beta beta'-subunits of Escherichia coli RNA-polymerase

The rpoC1 ts mutation affecting the RNA polymerase beta' subunit accelerates synthesis of RNA polymerase beta beta' subunits at 42 degrees C, while the surplus amount of subunits degrades in an hour's time. In a Ts strain with two RNA polymerase mutations, rpoC1 and rpoB251, we obtained a ts+ reversion designated opr24 which slows down degradation of surplus beta beta' subunits. The slowing down of degradation and the resulting accumulation of beta beta' subunits does not affect the kinetics of beta beta' subunit synthesis after the transfer to 42 degrees C. The effects of the opr24 are allele non-specific. The mutation also slows down degradation of beta' subunit and the amber fragment of beta subunit in the strain with subunit amber mutation rpoB22. Besides, the opr24 mutation reduces proteolysis of anomalous proteins containing canavanine. The opr24 mutation has been mapped between 17 and 21 minutes on the Escherichia coli map.     

Autogenous regulation of RNA polymerase beta subunit synthesis in vitro.

The effects of Escherichia coli RNA polymerase and its subassemblies and subunits on the in vitro synthesis of beta subunit directed by DNA from a lambda transducing phage lambdadrif+-6 were investigated. This phage carries the structural gene (rpoB) for beta subunit as well as the genes for EF (translation elongation factor)-Tu, some ribosomal proteins, and stable RNAs of the E. coli chromosome. Among the RNA polymerase proteins examined, the two oligomers, holoenzyme and alpha2beta complex, repressed the synthesis of only the beta subunit but not of other proteins encoded by the phage DNA. The results indicate that the expression of at least the betabeta' (rpoBC) operon is under autogenous regulation, in which both holoenzyme and alpha2beta complex function as regulatory molecules with repressor activity.     

Cloning and characterization of RNA polymerase core subunits of Chlamydia trachomatis by using the polymerase chain reaction.

Taking advantage of sequence conservation of portions of the alpha, beta, and beta' subunits of RNA polymerase of bacteria and plant chloroplasts, we have designed degenerate oligonucleotides corresponding to these domains and used these synthetic DNA sequences as primers in a polymerase chain reaction to amplify DNA sequences from the chlamydial genome. The polymerase chain reaction products were used as a probe to recover the genomic fragments encoding the beta subunit and the 5' portion of the beta' subunit from a library of cloned murine Chlamydia trachomatis DNA. Similar attempts to recover the alpha subunit were unsuccessful. Sequence analysis demonstrated that the beta subunit of RNA polymerase was located between genes encoding the L7/L12 ribosomal protein and the beta' subunit of RNA polymerase; this organization is reminiscent of the rpoBC operon of Escherichia coli. The C. trachomatis beta subunit overproduced in E. coli was used as an antigen in rabbits to make a polyclonal antibody to this subunit. Although this polyclonal antibody specifically immunoprecipitated the beta subunit from Chlamydia-infected cells, it did not immunoprecipitate core or holoenzyme. Immunoblots with this antibody demonstrated that the beta subunit appeared early in infection.     

Helicobacter pylori with separate beta- and beta'-subunits of RNA polymerase is viable and can colonize conventional mice

The genes encoding the beta- and beta'-subunits of RNA polymerase (rpoB and rpoC respectively) are fused as one continuous open reading frame in Helicobacter pylori and in other members of this genus, but are separate in other bacterial taxonomic groups, including the closely related genus Campylobacter. To test whether this beta-beta' tethering is essential, we used polymerase chain reaction-based cloning to separate the rpoB and rpoC moieties of the H. pylori rpoB-rpoC fusion gene with a non-polar chloramphenicol resistance cassette containing a new translational start, and introduced this construct into H. pylori by electro-transformation. H. pylori containing these separated rpoB and rpoC genes in place of the native fusion gene produced non-tethered beta and beta' RNAP subunits, grew well in culture and colonized and proliferated well in conventional C57BL/6 mice. Thus, the extraordinary beta-beta' tethering is not essential for H. pylori viability and gastric colonization.