A mutation in the RNA polymerase β′ subunit causing depressed ribosomal RNA synthesis in Escherichia coli

Molecular Genetics and Genomics - Macromolecular synthesis in an Escherichia coli mutant with a temperature-sensitive β′ subunit of RNA polymerase was analysed. At the non-permissive...     

X-ray Crystal Structure of Escherichia coli RNA Polymerase σ70 Holoenzyme

Escherichia coli RNA polymerase (RNAP) is the most studied bacterial RNAP and has been used as the model RNAP for screening and evaluating potential RNAP-targeting antibiotics. However, the x-ray crystal structure of E. coli RNAP has been limited to individual domains. Here, I report the x-ray structure of the E. coli RNAP σ⁷⁰ holoenzyme, which shows σ region 1.1 (σ_1.1) and the α subunit C-terminal domain for the first time in the context of an intact RNAP. σ_1.1 is positioned at the RNAP DNA-binding channel and completely blocks DNA entry to the RNAP active site. The structure reveals that σ_1.1 contains a basic patch on its surface, which may play an important role in DNA interaction to facilitate open promoter complex formation. The α subunit C-terminal domain is positioned next to σ domain 4 with a fully stretched linker between the N- and C-terminal domains. E. coli RNAP crystals can be prepared from a convenient overexpression system, allowing further structural studies of bacterial RNAP mutants, including functionally deficient and antibiotic-resistant RNAPs.     

Altered synthesis and stability of RNA polymerase holoenzyme subunits in mutants of Escherichia coli with mutations in the β or β′ subunit genes

Summary Bacteria with specific temperature sensitive lethal mutations in the gene for the subunit of RNA polymerase synthesize both the and subunits at a several fold higher rate at 42°C than wildtype cells relative to total protein. Synthesis of the and subunits proceeds at essentially the wild-type rates under these conditions. In contrast, a mutant with a temperature sensitive lethal mutation in the subunit gene synthesizes and at 42°C at slightly lower rates than wild-type, while and synthesis is not significantly altered. In all of the mutants at 42°C, newly synthesized subunits are stable, while the , and subunits are rapidly degraded. The apparent uncoupling of from subunit synthesis seen in the mutants at 42°C might suggest that the synthesis of these subunits is at least in part controlled by different mechanisms.     

Interaction of the lacZ β-galactosidase of Escherichia coli with some β-d-galactopyranoside competitive inhibitors

1. The location of the bivalent metal cation with respect to bound competitive inhibitors in Escherichia coli (lacZ) beta-galactosidase was investigated by proton magnetic resonance. 2. Replacement of Mg(2+) by Mn(2+) enhances both longitudinal and transverse relaxation of the methyl groups of the beta-d-galactopyranosyltrimethylammonium ion, and of methyl 1-thio-beta-d-galactopyranoside; linewidths are narrowed by increasing temperature. 3. The Mn(2+) ion is located 8-9A (0.8-0.9nm) from the centroid of the trimethylammonium group and 9A (0.9nm) from the average position of the methylthio protons. 4. The effective charge at the active site was probed by measurement of competitive inhibition constants (K(i) (o) and K(i) (+) respectively) for the isosteric ligands, beta-d-galactopyranosylbenzene and the beta-d-galactopyranosylpyridinium ion. 5. The ratio of inhibition constants (Q=K(i) (+)/K(i) (o)) obtained with 2-(beta-d-galactopyranosyl)-naphthalene and the beta-d-galactopyranosylisoquinolinium ion at pH7 with Mg(2+)-enzyme was identical, within experimental error, with that obtained with the monocyclic compounds. 6. The variation of Q for Mg(2+)-enzyme can be described by Q=0.1(1+[H(+)]/4.17x10(-10))/1+[H(+)]/10(-8)). 7. This, in the theoretical form for a single ionizable group, is ascribed to the ionization of the phenolic hydroxy group of tyrosine-501. 8. The variation of Q for Mg(2+)-free enzyme is complex, probably because of deprotonation of the groups normally attached to Mg(2+) as well as tyrosine-501.