A temperature-sensitive mutant of Escherichia coli with an altered RNA polymerase beta' subunit.

$\text{Penicillium charlesii}$ extracts contain UDP-galactose:NAD⁺ 2-hexosyl oxidoreductase (1). ADP-ribose also serves as a substrate resulting in formation of NADH and an oxidized ADP-ribose derivative. Treatment of the oxidized product with NaBH₄ followed by hydrolysis at pH 2 and 100° releases xylose as well as ribose. We conclude that ADP-D-$\text{glycero}$-D-$\text{glycero}$-3-pentosulose (ADP-3-ketoribose) is the product derived from ADP-ribose.     

Cyanobacterial RNA polymerase genes rpoC1 and rpoC2 correspond to rpoC of Escherichia coli.

The DNA-dependent RNA polymerase (ribonucleoside triphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) of cyanobacteria contains a unique core component, gamma, which is absent from the RNA polymerases of other eubacteria (G. J. Schneider, N. E. Tumer, C. Richaud, G. Borbely, and R. Haselkorn, J. Biol. Chem. 262:14633-14639, 1987). We present the complete nucleotide sequence of rpoC1, the gene encoding the gamma subunit, from the heterocystous cyanobacterium Nostoc commune UTEX 584. The derived amino acid sequence of gamma (621 residues) corresponds with the amino-terminal portion of the beta' polypeptide of Escherichia coli RNA polymerase. A second gene in N. commune UTEX 584, rpoC2, encodes a protein which shows correspondence with the carboxy-terminal portion of the E. coli beta' subunit. The rpoBC1C2 genes of N. commune UTEX 584 are present in single copies and are arranged in the order rpoBC1C2, and the coding regions are separated by short AT-rich spacer regions which have the potential to form very stable secondary structures. Our data indicate the occurrence of divergent evolution of structure in the eubacterial DNA-dependent RNA polymerase.     

Characterization and epitope mapping of monoclonal antibodies directed against the beta' subunit of the Escherichia coli RNA polymerase.

Monoclonal antibodies (mAbs) raised against the beta' subunit of the Escherichia coli RNA polymerase were used to probe the structure and function of this subunit. Of the five anti-beta' monoclonal antibodies studied, only mAb 311G2 is a strong inhibitor of RNA polymerase activity. This antibody binds to an epitope which is exposed in both the assembled holoenzyme and isolated beta' subunit. In contrast, the null antibodies bind to the free beta' subunit but very weakly to native RNA polymerase. It would appear that the beta' domain in which their epitopes reside is either conformationally altered or blocked due to interaction with other subunits in native RNA polymerase. In order to locate the positions of the epitopes for these five monoclonal antibodies, a series of overlapping deletion mutants have been constructed by partial restriction and religation of the beta' gene present in pT7 beta' (Zalenskaya, K., Lee, J., Gujuluva, C. N., Shin, Y. K., Slutsky, M., nd Goldfarb, A. (1990) Gene 89, 7-12). The presence of the epitopes for each of the anti-beta' monoclonal antibodies was assessed by Western blotting. The results indicate that the epitopes for mAb 340F11, mAb 370F3, mAb 371D6, and mAb 372B2 are located between amino acids 817-876. This region may be important in enzyme assembly or subunit-subunit interaction. The epitope for the inhibitory antibody, mAb 311G2, is located between amino acids 1047-1093. This region may be involved in the catalytic function of RNA polymerase.     

Pea chloroplast DNA encodes homologues of Escherichia coli ribosomal subunit S2 and the beta''-subunit of RNA polymerase.

The nucleotide sequence has been determined of a segment of 4680 bases of the pea chloroplast genome. It adjoins a sequence described elsewhere that encodes subunits of the F0 membrane domain of the ATP-synthase complex. The sequence contains a potential gene encoding a protein which is strongly related to the S2 polypeptide of Escherichia coli ribosomes. It also encodes an incomplete protein which contains segments that are homologous to the beta'-subunit of E. coli RNA polymerase and to yeast RNA polymerases II and III.     

Biosynthesis of the beta and beta' subunits of RNA polymerase in Escherichia coli

The amounts of the β and β′ subunits of the DNA-dependent RNA polymerase relative to the amount of total protein synthesized have been determined under a number of growth conditions in two strains of Escherichia coli. The results of these measurements have been expressed as the relative rate of synthesis of core RNA polymerase, α_p, assuming the four constituent subunits (2α, 1β and 1β′) to be synthesized in equivalent amounts.This quantity, α_p, was found not to vary greatly with the growth rate μ. For glucose-grown cells of E. coli B/r (μ = 1.5 doublings/h) α_p = 1.4%, corresponding to about 7000 molecules of core RNA polymerase per cell. For slowgrowing cells the value obtained for α_p is lower and for fast-growing cells somewhat 3 higher. The comparison of these values with the number of RNA polymerase molecules estimated to be actively engaged in RNA synthesis indicates that both slow- and fast-growing cells contain a surplus of RNA polymerase, if the catalytic unit is assumed to be the monomer of core RNA polymerase.In addition to the measurements of cells during balanced growth at various rates, α_p has been determined during the transition from one growth rate to another and during synchronous growth. During a shift-up the rate of synthesis of polymerase follows closely the rate of total protein synthesis, α_p being nearly constant for a period of twenty minutes after the shift. In a synchronously dividing culture of E. coli B/r, α_p was seen to be fairly constant during two cycles of synchronous division. It appears that α_p is rather insensitive to the effect of gene doubling during the cell cycle.     

Localization of Escherichia coli rpoC mutations that affect RNA polymerase assembly and activity at high temperature

We localized five rpoC (beta') mutations affecting Escherichia coli RNA polymerase assembly. The Ts4, XH56, and R120 mutations changed beta' residues conserved throughout eubacteria; the JE10092 mutation occurred in the hypervariable region; rpoC1 (TsX) changed a universally conserved residue and corresponds to yeast rpb1-1. Thus, distinct, predominantly conserved beta' residues participate in interactions holding RNA polymerase together.     

Escherichia coli RNA polymerase subunit omega and its N-terminal domain bind full-length beta' to facilitate incorporation into the alpha2beta subassembly.

The omega subunit of Escherichia coli RNA polymerase, consisting of 90 amino acids, is present in stoichiometric amounts per molecule of core RNA polymerase (alpha2betabeta'). The presence of omega is necessary to restore denatured RNA polymerase in vitro to its fully functional form, and, in an omega-less strain of E. coli, GroEL appears to substitute for omega in the maturation of RNA polymerase. The X-ray structure of Thermus aquaticus core RNA polymerase suggests that two regions of omega latch on to beta' at its N-terminus and C-terminus. We show here that omega binds only the intact beta' subunit and not the beta' N-terminal domain or beta' C-terminal domain, implying that omega binding requires both these regions of beta'. We further show that omega can prevent the aggregation of beta' during its renaturation in vitro and that a V8-protease-resistant 52-amino-acid-long N-terminal domain of omega is sufficient for binding and renaturation of beta'. CD and functional assays show that this N-terminal fragment retains the structure of native omega and is able to enhance the reconstitution of core RNA polymerase. Reconstitution of core RNA polymerase from its individual subunits proceeds according to the steps alpha + alpha --> alpha2 + beta --> alpha2beta + beta' --> alpha2betabeta'. It is shown here that omega participates during the last stage of enzyme assembly when beta' associates with the alpha2beta subassembly.     

Genetic suppression of a temperature-sensitive groES mutation by an altered subunit of RNA polymerase of Escherichia coli K-12.

Temperature-resistant suppressor mutants were isolated from Escherichia coli mutant strain groES131(Ts). Phage P1-mediated transduction and a two-dimensional gel electrophoretic analysis of cellular proteins indicated that these suppressor mutants carry an additional mutation in either the groEL gene or the rpoA gene.