Small cytoplasmic RNA of Bacillus subtilis: functional relationship with human signal recognition particle 7S RNA and Escherichia coli 4.5S RNA.

Small cytoplasmic RNA (scRNA; 271 nucleotides) is an abundant and stable RNA of the gram-positive bacterium Bacillus subtilis. To investigate the function of scRNA in B. subtilis cells, we developed a strain that is dependent on isopropyl-beta-D-thiogalactopyranoside for scRNA synthesis by fusing the chromosomal scr locus with the spac-1 promoter by homologous recombination. Depletion of the inducer leads to a loss of scRNA synthesis, defects in protein synthesis and production of alpha-amylase and beta-lactamase, and eventual cell death. The loss of the scRNA gene in B. subtilis can be complemented by the introduction of human signal recognition particle 7S RNA, which is considered to be involved in protein transport, or Escherichia coli 4.5S RNA. These results provide further evidence for a functional relationship between B. subtilis scRNA, human signal recognition particle 7S RNA, and E. coli 4.5S RNA.     

Characterization of conserved bases in 4.5S RNA of Escherichia coli by construction of new F' factors

To more clearly understand the function of conserved bases of 4.5S RNA, the product of the essential ffs gene of Escherichia coli, and to address conflicting results reported in other studies, we have developed a new genetic system to characterize ffs mutants. Multiple ffs alleles were generated by altering positions that correspond to the region of the RNA molecule that interacts directly with Ffh in assembly of the signal recognition particle. To facilitate characterization of the ffs mutations with minimal manipulation, recombineering was used to construct new F' factors to easily move each allele into different genetic backgrounds for expression in single copy. In combination with plasmids that expressed ffs in multiple copy numbers, the F' factors provided an accurate assessment of the ability of the different 4.5S RNA mutants to function in vivo. Consistent with structural analysis of the signal recognition particle (SRP), highly conserved bases in 4.5S RNA are important for binding Ffh. Despite the high degree of conservation, however, only a single base (C62) was indispensable for RNA function under all conditions tested. To quantify the interaction between 4.5S RNA and Ffh, an assay was developed to measure the ability of mutant 4.5S RNA molecules to copurify with Ffh. Defects in Ffh binding correlated with loss of SRP-dependent protein localization. Real-time quantitative PCR was also used to measure the levels of wild-type and mutant 4.5S RNA expressed in vivo. These results clarify inconsistencies from prior studies and yielded a convenient method to study the function of multiple alleles.     

Mutations in the gene for EF-G reduce the requirement for 4.5S RNA in the growth of E. coli

A general strategy is described for the isolation of suppressors of essential genes whose functions are unknown. This strategy was used to analyze the role of 4.5S RNA, an essential RNA of E. coli. In this strategy, the structural gene for 4.5S RNA is fused to the Ptac promoter in such a way that the strain becomes dependent upon inducers of lac for growth. Mutants mapping to fus, the structural gene for protein synthesis elongation factor G, appear as spontaneous, inducer-independent revertants. These mutants alter the intracellular distribution of 4.5S RNA such that it sediments at 70S or greater. Furthermore, the increased sedimentation velocity is sensitive to the antibiotic puromycin. These results show that 4.5S RNA physically associates with the ribosome in performing its essential function, and that this association is mediated by elongation factor G.     

4.5S RNA: does form predict function?

4.5S RNA is a stable RNA of Escherichia coli, and functional homologs of the molecule apparently exist in all prokaryotes: eubacteria, archebacteria, and mycoplasma. Genetic and physiological measurements of the function of 4.5S RNA in E. coli indicate a role for this RNA in protein synthesis. A conserved domain of 4.5S RNA displays structural similarity with the eukaryotic 7S RNA that functions in protein secretion. Although complementation by eukaryotic 7S RNAs remains to be demonstrated, a number of archaebacterial 7S RNAs are able to replace 4.5S RNA for growth of E. coli, and 4.5S RNA is able to mediate a number of 7S RNA functions in vitro. Surprisingly, no effects on protein secretion in E. coli have been directly attributed to 4.5S RNA. These observations raise the question of whether molecules of similar structure necessarily perform the same function.     

Genetic Screen Yields Mutations in Genes Encoding All Known Components of the Escherichia coli Signal Recognition Particle Pathway

We describe the further utilization of a genetic screen that identifies mutations defective in the assembly of proteins into the Escherichia coli cytoplasmic membrane. The screen yielded mutations in each of the known genes encoding components of the E. coli signal recognition particle pathway: ffh, ffs, and ftsY, which encode Ffh, 4.5S RNA, and FtsY, respectively. In addition, the screen yielded mutations in secM, which is involved in regulating levels of the SecA component of the bacterium's protein export pathway. We used a sensitive assay involving biotinylation to show that all of the mutations caused defects in the membrane insertions of three topologically distinct membrane proteins, AcrB, MalF, and FtsQ. Among the mutations that resulted in membrane protein insertion defects, only the secM mutations also showed defects in the translocation of proteins into the E. coli periplasm. Genetic evidence suggests that the S382T alteration of Ffh affects the interaction between Ffh and 4.5S RNA.     

Regulated high-level expression of the Herpes simplex type I thymidine kinase gene in Escherichia coli

A plasmid-borne Herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) gene (tk) was expressed in Escherichia coli by inserting a 203-bp lacL8/UV5 promoter-operator segment, in frame, 53 bp 5' to the native tk translational start codon. The hybrid gene created by this fusion encodes a polypeptide which has 25 additional amino acids on the amino terminus of the HSV-1 TK protein and phenotypically complements a tdk- mutation of E. coli. This fusion polypeptide has been characterized by maxicell, immunoprecipitation, and native gel techniques, and its activity is inhibited by anti-HSV-1 antibody. In a tk expressor strain containing a F' lacIq (which overproduces the lactose repressor), the isopropyl-beta-D-thiogalactoside (IPTG) causes greater than 1000-fold coordinate induction of the plasmid-encoded TK and chromosomal beta-galactosidase activities. Pulse-chase induction demonstrates the fused TK polypeptide to be as stable as beta-galactosidase. HSV-1 tk-specific RNA isolated from this bacterial strain has a short half-life characteristic of bacterial messages.     

Loss of 4.5S RNA induces the heat shock response and lambda prophage in Escherichia coli.

During depletion of 4.5S RNA, cells of Escherichia coli displayed a heat shock response that was simultaneous with the first detectable effect on ribosome function and before major effects on cell growth. Either 4.5S RNA is involved directly in regulating the heat shock response, or this particular impairment of protein synthesis uniquely induces the heat shock response. Several hours later, lambda prophage was induced and the cells lysed.     

Protein Z is not an endonuclease of the recF recombination pathway in Escherichia coli K12

A 74 kD protein was extracted from Escherichia coli cells and purified under the physiological conditions. The protein is able to catalyze the reactions of endonucleolytic degradation of plasmid DNA. The genetic determinant coding for the 74 KD protein synthesis has been localized between 17 and 27 min on Escherichia coli chromosomal map. The endonuclease previously described as a recF gene dependent "protein Z" (Krivonogov S. V., Novitskaja V. A. Mol. Gen. Genet., 1982, v, 187, p. 302) is shown to be independent of the integrity of Escherichia coli recF gene.     

Release of template restriction in chromatin by nuclear 4.5s RNA.

The stimulatory mechanism of RNA synthesis of calf-thymus chromatin by nuclear 4.5 S RNA from the homologous tissue was investigated by using exogenously added Escherichia coli RNA polymerase. The RNA synthesis was initiated at low concentration of salt, and then the chain elongation was achieved at high concentration of ammonium sulfate in the presence of polyvinyl sulfate. Under these conditions the number of binding sites of RNA polymerase on chromatin which were capable of initiating RNA chain was increased by the addition of the 4.5 S RNA. This stimulation was presumed to result from the release of template restriction in chromatin. The polyvinyl salt minimized ribonuclease activity without changing the RNA polymerase activity bound to the template. Neither rearrangement nor release of chromatin proteins affected the amount or size of RNA produced. Preliminary analysis suggested that the molecular species of RNA produced upon the addition of the 4.5 S RNA from various tissues seemed to be heterologous.     

Vaccinia virus morphogenesis is interrupted when expression of the gene encoding an 11-kilodalton phosphorylated protein is prevented by the Escherichia coli lac repressor.

A conditional lethal vaccinia virus mutant, which constitutively expresses the Escherichia coli lac repressor and has the lac operator controlling the F18R gene (the 18th open reading frame of the HindIII F fragment of the vaccinia virus strain WR genome) encoding an 11-kDa protein, was previously shown to be dependent on the inducer isopropyl-beta-D-thiogalactoside (IPTG) for replication (Y. Zhang and B. Moss, Proc. Natl. Acad. Sci. USA 88:1511-1515, 1991). Further studies indicated that the yield of infectious virus could be regulated by titration with IPTG and that virus production was arrested by IPTG removal at appropriate times. Under nonpermissive conditions, an 11-kDa protein reactive with antiserum raised to a previously described DNA-binding phosphoprotein (S. Y. Kao and W. R. Bauer, Virology 159:399-407, 1987) was not synthesized, indicating that the latter is the product of the F18R gene. In the absence of IPTG, replication of viral DNA and the subsequent resolution of concatemeric DNA molecules appeared normal. Omission of IPTG did not alter the kinetics of early and late viral protein synthesis, although the absence of the 11-kDa polypeptide was noted by labeling infected cells with [35S]methionine or [32P]phosphate. Pulse-chase experiments revealed that proteolytic processing of the major viral structural proteins, P4a and P4b, was inhibited under nonpermissive conditions, suggesting a block in virus maturation. Without addition of IPTG, the failure of virus particle formation was indicated by sucrose gradient centrifugation of infected cell lysates and by the absence of vaccinia virus-mediated pH-dependent cell fusion. Electron microscopic examination of infected cells revealed that immature virus particles, with aberrant internal structures, accumulated when synthesis of the 11-kDa DNA-binding protein was prevented.     

Escherichia coli 4.5S RNA gene function can be complemented by heterologous bacterial RNA genes.

The essential 4.5S RNA gene of Escherichia coli can be complemented by 4.5S RNA-like genes from three other eubacteria, including both gram-positive and gram-negative organisms. Two of the genes encode RNAs similar in size to the E. coli species; the third, from Bacillus subtilis, specifies an RNA more than twice as large. The heterologous genes are expressed efficiently in E. coli, and the product RNAs resemble those produced by cognate cells. We conclude that the heterologous RNAs can replace E. coli 4.5S RNA and that the essential function of 4.5S RNA is evolutionarily conserved. A consensus structure is presented for the functionally related 4.5S RNA homologs.