Competition of rifampicin with binding of substrate and RNA to RNA polymerase

The rate of formation of dinucleoside tetraphosphate, pppApU, from ATP and UTP by RNA polymerase on the A1 promoter of the mutant D111 of bacteriophage T7 is distinctly and specifically reduced not only by the third template-directed nucleotide, CTP, but also by CMP. The inhibitory effect of CMP is not changed when the enzyme contains prebound rifampicin. The synthesis of pppApU is also strongly reduced after preincubation of the enzyme with RNA. This inhibitory effect of RNA is, however, distinctly diminished by rifampicin bound to the enzyme prior to the addition of RNA. On the other hand RNA can suppress the specific binding of the antibiotic to the RNA polymerase subassembly alpha 2 beta.     

Monoclonal antibodies as probes of the topological arrangement of the alpha subunits of Escherichia coli RNA polymerase

Three monoclonal anti-alpha antibodies were used to study the properties of the alpha subunit of Escherichia coli RNA polymerase. None of the monoclonal antibodies inhibited the d(A-T)n-directed synthesis of r(A-U)n. Reassembly of the RNA polymerase core was blocked by mAb 129C4 or mAb 126C6 while no effect was observed with mAb 124D1. The conversion of premature to mature core was partially inhibited by mAb 129C4 and almost totally inhibited by mAb 126C6. The data suggest that during the course of core assembly at least one of the alpha subunits undergoes conformational changes. The increase in affinity of mAb 126C6 for assembled alpha compared with free alpha also implies that alpha undergoes conformational changes during RNA polymerase assembly. Double antibody binding studies showed that the epitopes for mAb 124D1 and mAb 129C4 are available on only one of the alpha subunits in RNA polymerase. It would appear that the relevant domain on one of the alpha subunits in RNA polymerase is well exposed whereas this domain on the second alpha subunit is shielded by interaction with regions of the large beta and beta' subunits. The alpha domain in which the epitope for mAb 126C6 resides is not impeded by subunit interactions in the RNA polymerase. The data obtained also suggest that in the holoenzyme the sigma subunit may be positioned close to one of the alpha subunits, probably to the more exposed alpha. The alpha beta complex is the minimal stable subassembly since one of the alpha subunits dissociates from the alpha 2 beta complex following binding of any of the monoclonal antibodies studied.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Reconstitution studies show that rifampicin resistance is determined by the largest polypeptide of Bacillus subtilis RNA polymerase.

A procedure has been developed to separate the subunits of Bacillus subtilis RNA polymerase rapidly and in good yield. The method involved the use of a blue dextran-Sepharose column which bound the beta' subunit. A phosphocellulose column was used to separate the alpha and beta subunits. During purification, the enzyme eluted from the DNA-cellulose column in three separate forms in the order alpha2betabeta'deltaomega1,alpha2betabeta'omega1, and alpha2betabeta'omega1sigma. Subunit reconstitution studies with RNA polymerase subunits from wild type and a rifampicin-resistant mutant indicated that the largest polypeptide was responsible for rifampicin resistance. Thus, this subunit is referred to as beta. The mobility of the subunits in sodium dodecyl sulfate-polyacrylamide gel electrophoresis cannot be used as the sole criterion for designating the functions of the subunits of RNA polymerase.     

Purification and partial characterization of DNA-dependent RNA polymerase from Rhodomicrobium vannielii

DNA-dependent RNA polymerase has been isolated from Rhodomicrobium vannielii. Like those from other eubacteria, the enzyme contained four subunits: beta and beta prime (Mr 155,000), alpha (Mr 38,000), and sigma (Mr 98,000). Analysis by isoelectric focussing showed that both alpha and sigma had several forms with different isoelectric pH values. The enzyme was sensitive to rifampicin (5 ng rifampicin ml-1 gave 50% inhibition) and capable of specific promoter selection with DNA from R. vannielii, calf thymus and phage T7D111.     

DNA-dependent RNA polymerase from Pseudomonas aeruginosa

DNA-dependent RNA polymerase was purified from Pseudomonas aeruginosa. The subunit structure was typical of other eubacterial RNA polymerases in having beta' (157,000), beta (148,000), sigma (87,000), and alpha 2 (45,000) subunits as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was dependent on Mg2+, displaying optimal activity at 10 mM MgCl2. Ca2+ and Zn2+ could not replace MgCl2 in the assay system, while Mn2+, produced partial activity. KCl at concentrations greater than 10 mM inhibited enzyme activity. Optimal enzyme activity was observed at pH 8.5-9.0. The RNA polymerase was stable in 50% (w/v) glycerol at 4 degrees C for more than 3 months. Enzyme activity was inhibited in vitro by heparin, streptolydigin, streptovaracin, actinomycin D, and rifampicin.     

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.     

Formation of a RNA polymerase sub-assembly composed of subunit alpha from Escherichia coli and of subunit beta from Micrococcus luteus.

Functionally equivalent subunits of RNA polymerase from Micrococcus luteus and Escherichia coli differ from each other in many molecular and antigenic properties. In spite of these differences, subunit alpha from E. coli and subunit beta from M. luteus form a complex alpha2beta, when incubated together. This complex binds rifampicin tightly, which the isolated subunits do not. The hybrid complex is very similar in its properties to the complex alpha2beta formed only from E. coli or M. luteus subunits. Since the sub-assembly alpha2beta from E. coli is reported to be an obligatory intermediate in the assembly process of complete RNA polymerase, the newly described hybrid sub-assembly may function similarly as an intermediate in the formation of the hybrid form of RNA polymerase described earlier.     

Solution structure and RNA interactions of the RNA recognition motif from eukaryotic translation initiation factor 4B

Eukaryotic initiation factor 4B (eIF4B) is a multidomain protein with a range of activities that serves primarily to promote association of messenger RNA to the 40S ribosomal subunit during translation initiation. We report here the solution structure of the eIF4B RNA recognition motif (RRM) domain. It adopts a classical RRM fold, with a beta alpha beta beta alpha beta topology. The most striking difference with other RRM structures is in the disposition of loop 3, which connects the beta 2 and beta 3 strands and is implicated in RNA recognition. This loop folds down against the body of the RRM and exhibits restricted motion on a milli- to microsecond time scale. Although it contributes to a large basic patch on the RNA binding surface, it does not protrude out from the domain as observed in other RRM structures, possibly implying a different mode of RNA binding. On its own, the core RRM domain provides only a relative weak interaction with RNA targets and appears to require extensions at the N- and C-terminus for high-affinity binding.     

Early and late functions in a bipartite RNA virus: evidence for translational control by competition between viral mRNAs.

It has been shown previously that Drosophila cells infected with black beetle virus synthesize an early viral protein, protein A, a putative element of the viral RNA polymerase. Synthesis of protein A declines sharply by 6 h postinfection, whereas synthesis of viral coat protein alpha continues for at least 14 h. The early shutoff in protein A synthesis occurred despite the presence of equimolar proportions of the mRNAs for proteins A and alpha, RNAs 1 and 2, respectively. We have now been able to mimic this translational discrimination in a cell-free protein-synthesizing system prepared from infected or uninfected Drosophila cells, thus allowing further analysis of the mechanism by which translation of RNA 1 is selectively turned off. The results revealed no evidence for control by virus-encoded proteins or by virus-induced modification of mRNAs by the cell-free system. Rather, with increasing RNA concentration, viral RNA 1 was outcompeted by its genomic partner, RNA 2. This suggests that the early shutoff in intracellular synthesis of protein A is due to decreasing ability of RNA 1 to compete for a rate-controlling translational factor(s) as the concentration of viral RNAs accumulates within the infected cell.     

The RNA-dependent DNA polymerase of avian sarcoma virus B77. Binding of viral and nonviral ribonucleic acids to the alpha, beta2, and alphabeta forms of the enzyme.

The extent of binding of various RNA species to the three forms of avian sarcoma virus B77 RNA-dependent DNA polymerase was determined using a sensitive nitrocellulose filter binding technique which was capable of detecting binding reactions with association constants as low as 3 X 10(6) liters X mole-1. All three enzyme forms, alphabeta, beta2, and alpha, bound to all single-stranded RNA species that were tested, including nonviral RNAs. 70 S viral RNA exhibited the highest association constant (about 10(11) liters X mole-1), and a population of virus-derived tRNA molecules from which tRNATrp had been removed, the lowest (about 3000 times lower). The affinity for other RNAs was roughly proportional to their size. The affinity of RNAs for the alphabeta enzyme form always exceeded that for the two others by a factor that depended on the particular RNA, never exceeded 6 and was sometimes as low as 1.2. The association constant of the alphabeta enzyme form with viral 70 S RNA was about 15-fold higher than that with viral 35 S RNA. 35 S RNA annealed to tRNATrp had an association constant that was only 2.5 times higher than that of 35 S RNA alone. This finding suggests that the tertiary structure of 70 S RNA plays a significant role in its affinity for B77 DNA polymerase.     

The role of Halobacterium cutirubrum deoxyribonucleic acid-dependent ribonucleic acid polymerase subunits in initiation and polymerization

1. The two subunits alpha and beta of Halobacterium cutirubrum DNA-dependent RNA polymerase are required in equimolar amounts for RNA synthesis to occur in vitro at the maximum rate. 2. In the absence of bivalent cations no interaction occurs between alpha and beta subunits or between the subunits and DNA. 3. Mn(2+) causes the subunits to form a 1:1 complex that still does not bind to the template. 4. Mg(2+) permits binding of the Mn(2+)-mediated complex to DNA. 5. The complete enzyme, alphabeta, is inhibited by rifampicin and only the beta subunit relieves the inhibition when added in excess. 6. Rifampicin-insensitive, template-dependent RNA synthesis occurs in the presence of protein alpha alone provided an oligonucleotide with a 5'-purine terminus is supplied as primer. 7. In the primed reaction with the alpha protein and an oligonucleotide, the template specificity is independent of the ionic strength, in contrast with the marked effect of salt concentration on the template specificity of the complete enzyme. 8. It is concluded that the beta protein controls the specificity of chain initiation and the template specificity of the complete enzyme and also carries the rifampicin-binding site, whereas the catalytic site is on the alpha subunit.     

Replication of nodamura virus after transfection of viral RNA into mammalian cells in culture.

Nodamura virus (NOV) was purified from the hind limbs of infected suckling mice and used as a source of the two genomic RNAs of the virus, RNA 1 and RNA 2. Upon transfection of the viral RNAs into baby hamster kidney (BHK21) cells in culture, vigorous RNA replication ensued and single-stranded RNAs 1 and 2 accumulated to reach an abundance which approximated that of the cellular rRNAs. Transient synthesis of a small subgenomic RNA (RNA 3) was also observed, and double-stranded versions of RNAs 1, 2, and 3 were detected. Three major viral proteins were synthesized in transfected cells. Protein A (about 115 kDa) and protein B (about 15 kDa) were made transiently at early times after transfection, whereas a large amount of protein alpha (43 kDa), the precursor to the two viral coat proteins, was made continuously starting later in the infectious cycle. When very low concentrations of viral RNAs were used for transfection, preferential replication of RNA 1 occurred. This result was attributed to segregation of the transfected viral RNAs to separate cells in culture and the subsequent replication and amplification of RNA 1 in cells that had received no RNA 2. Accordingly, multiple passages of the viral RNAs by transfection at the limit dilution resulted in the purification of RNA 1 free of RNA 2 and demonstrated that RNA 1 was capable of prolonged autonomous replication which was also accompanied by the continuous synthesis of RNA 3. In cells transfected with RNA 1 alone, protein alpha was not synthesized and proteins A and B were made continuously. Electron microscopic analysis of BHK21 cells 24 h after transfection with NOV RNAs 1 and 2 showed that large numbers of virus particles accumulated in the cytoplasm and formed paracrystalline arrays in some regions. Whole NOV purified from transfected BHK21 cells was infectious for suckling mice and had an electrophoretic mobility that was similar but not identical to that of NOV purified from infected mouse muscle. The high yield of NOV, its simple genetic composition, and its unusual genome strategy make this virus an attractive system for the study of viral RNA replication in animal cells.