Time of action of 4.5 S RNA in Escherichia coli translation

A new class of suppressor mutants helps to define the role of 4.5 S RNA in translation. The suppressors reduce the requirement for 4.5 S RNA by increasing the intracellular concentration of uncharged tRNA. Suppression probably occurs by prolonging the period in which translating ribosomes have translocated but not yet released the uncharged tRNA, indicating that this is the point at which 4.5 S RNA enters translation. The release of 4.5 S RNA from polysomes is affected by antibiotics that inhibit protein synthesis. The antibiotic-sensitivity of this release indicates that 4.5 S RNA exits the ribosome following translocation and prior to release of protein synthesis elongation factor G. These results indicate that 4.5 S RNA acts immediately after ribosomal translocation. A model is proposed in which 4.5 S RNA stabilizes the post-translocation state by replacing 23 S ribosomal RNA as a binding site for elongation factor G. The 4.5 S RNA-requirement of mutants altered in 23 S ribosomal RNA support this model.     

Effect of 4.5S RNA depletion on Escherichia coli protein synthesis and secretion.

We examined the synthesis of individual proteins following depletion of 4.5S RNA by using a strain deficient in the induction of heat shock proteins. We found that initially the synthesis of all proteins was equally affected, and the peptide elongation rate was reduced by approximately 10%. For up to 1 generation time after the onset of inhibition of total protein synthesis, the processing of secreted proteins was unaffected. After further depletion of 4.5S RNA, accumulation of precursors of secreted proteins was observed under some growth conditions.     

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.     

Analysis of Escherichia coli 4.5S RNA binding affinity to Ffh and EF-G

Escherichia coli 4.5S RNA is a member of the signal recognition particle RNA family that binds to Ffh and EF-G proteins in vivo. To assess the binding affinity of E. coli 4.5S RNA, wild-type Ffh and a series of amino terminal truncated EF-G mutants with a histidine tag were over-expressed in Escherichia coli and purified. Among them, EF-G mutants with a deletion of all upstream sequences up to and including the second or the third GTP binding sequence element were expressed at high levels and bound with the same activity as wild-type EF-G. Nitrocellulose filter binding assays revealed that the binding affinity values (M(1/2)) for Ffh and EF-G, defined as the concentration giving half-maximal binding, were 0.15 microM and 1.5 microM, respectively. Moreover, we also show that very little EF-G can form a stable complex with 4.5S RNA in vivo, whereas almost all Ffh binds to 4.5S RNA.     

Genetic selection and DNA sequences of 4.5S RNA homologs.

A general strategy for cloning the functional homologs of an Escherichia coli gene was used to clone homologs of 4.5S RNA from other bacteria. The genes encoding these homologs were selected by their ability to complement a deletion of the gene for 4.5S RNA. DNA sequences of the regions encoding the homologs were determined. Since this approach does not require that the homologous genes hybridize with probes generated from the E. coli sequence, the sequences of the homologs were not all similar to the sequence of the E. coli gene. Despite the dissimilarity of the primary sequences of some of the homologs, all could be folded to obtain a similar structure.     

Decreased requirement for 4.5S RNA in 16S and 23S rRNA mutants of Escherichia coli

4.5S RNA is the bacterial homolog of the mammalian signal recognition particle (SRP) RNA that targets ribosome-bound nascent peptides to the endoplasmic reticulum. To explore the interaction of bacterial SRP with the ribosome, we have isolated rRNA suppressor mutations in Escherichia coli that decrease the requirement for 4.5S RNA. Mutations at C732 in 16S rRNA and at A1668 and G1423 in 23S rRNA altered the cellular responses to decreases in both Ffh (the bacterial homolog of SRP54) and 4.5S RNA levels, while the C1066U mutation in 16S rRNA and G424A mutation in 23S rRNA affected the requirement for 4.5S RNA only. These data are consistent with a dual role for 4.5S RNA, one involving co-translational protein secretion by a 4.5S-Ffh complex, the other involving free 4.5S RNA.     

The 4.5 S RNA gene of Escherichia coli is essential for cell growth

The Escherichia coli gene coding for the metabolically stable 4.5 S RNA (ffs) has been shown to be required for cell viability. Essentiality was demonstrated by examining the recombination behavior of substitution mutations of ffs generated in vitro. Substitution mutants of ffs are able to replace the chromosomal allele only in the presence of a second, intact copy of ffs. Independent evidence of essentiality and the finding that 4.5 S RNA is important for protein synthetic activity came from characterization of cells dependent on the lac operon inducer isopropyl-beta-D-thiogalactoside for ffs gene expression. Here, a strain dependent on isopropyl-beta-D-thiogalactoside for 4.5 S RNA synthesis was developed by inactivation of the chromosomal ffs allele and lysogenization by a lambda phage containing 4.5 S DNA fused to a hybrid trp-lac promoter. Withdrawal of the thiogalactoside leads to a deficiency in 4.5 S RNA, a dramatic loss in protein synthesis activity, and eventual cell death. Tagging of the chromosomal ffs region with a kanamycin-resistance gene allowed mapping of the 4.5 S RNA gene. Results from this analysis place ffs near lon at approximately ten minutes on the E. coli linkage map.     

Concentrations of 4.5S RNA and Ffh protein in Escherichia coli: the stability of Ffh protein is dependent on the concentration of 4.5S RNA.

We measured the concentrations of both 4.5S RNA and Ffh protein under a variety of growth conditions and found that there were 400 molecules of 4.5S RNA per 10,000 ribosomes in wild-type cells and that the concentration of Ffh protein was one-fourth of that. This difference in concentration is 1 order of magnitude less than that previously reported but still significant. Pulse-chase labeling experiments indicated that Ffh protein is unstable in cells carrying ffh on high-copy-number plasmids and that simultaneous overproduction of 4.5S RNA stabilizes Ffh protein. Our analyses show that free Ffh protein is degraded with a half-life of approximately 20 min. We also tested whether three previously isolated suppressors of 4.5S RNA deficiency could reduce the requirement for Ffh protein. Since the two sffE suppressors do not suppress the Ffh requirement, we suggest that 4.5S RNA either acts in a sequential reaction with Ffh or has two functions.     

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.     

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.     

Genes for 7S RNAs can replace the gene for 4.5S RNA in growth of Escherichia coli.

4.5S RNAs of eubacteria and 7S RNAs of archaebacteria and eukaryotes exist in a hairpin conformation. The apex of this hairpin displays structural and sequence similarities among both 4.5S and 7S RNAs. Furthermore, a hyphenated sequence of 16 nucleotides is conserved in all eubacterial 4.5S RNAs examined. In this article I report that 7S RNAs that contain this 16-nucleotide sequence are able to replace 4.5S RNAs and permit growth of Escherichia coli.     

Minimal functional structure of Escherichia coli 4.5 S RNA required for binding to elongation factor G

Escherichia coli cells contain abundant amounts of metabolically stable 4.5 S RNA. Consisting of 114 nucleotides, 4.5 S RNA is structurally homologous to mammalian 7 S RNA, and it plays an essential role in targeting proteins containing signal peptide to the secretory apparatus by forming an signal recognition-like particle with Ffh protein. It also binds independently to protein elongation factor G (EF-G) and functions in the translation process. This RNA contains a phylogenetically conserved RNA domain, the predicted secondary structure of which consists of a hairpin motif with two bulges. We examined the binding activity of mutants with systematic deletions to define the minimal functional interaction domain of 4.5 S RNA that interacts with EF-G. This domain consisted of 35-nucleotides extending from 36 to 70 nucleotides of mature 4.5 S RNA and contained two conserved bulges in which mutations of A47, A60, G61, C62, A63, and A67 diminished binding to EF-G, whereas those at A39, C40, C41, A42, G48, and G49 did not affect binding. These data suggested that the 10 nucleotides in 4.5 S RNA, which are conserved between 4.5 S RNA and 23 S rRNA, have a key role for EF-G binding. Based on the NMR-derived structure of mutant A47U, we further verified that substituting U at A47 causes striking structural changes and the loss of the symmetrical bulge. These results indicate the mechanism by which EF-G interacts with 4.5 S RNA and the importance of the bulge structure for EF-G binding.     

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.     

Structure of 4.5S RNA in the signal recognition particle of Escherichia coli as studied by enzymatic and chemical probing.

The structure of 4.5S RNA, the Escherichia coli homologue of the signal recognition particle (SRP) RNA, alone and in the SRP complex with protein P48 (Ffh) was probed both enzymatically and chemically. The molecule is largely resistant against single strand-specific nucleases, indicating a highly base paired structure. Reactivity appears mainly in the apical tetraloop and in one of the conserved internal loops. Although some residues are found reactive toward dimethylsulphate and kethoxal in regions predicted to be unpaired by the phylogenetic secondary structure model of 4.5S RNA, generally the reactivity is low, and some residues in internal loops are not reactive at all. RNase V1 cleaves the RNA at multiple sites that coincide with predicted helices, although the cleavages show a pronounced asymmetry. The binding of protein P48 to 4.5S RNA results in a protection of residues in the apical part of the molecule homologous to eukaryotic SRP RNA (domain IV), whereas the cleavages in the conserved apical tetraloop are not protected. Hydroxyl radical treatment reveals an asymmetric pattern of backbone reactivity; in particular, the region encompassing nucleotides 60-82, i.e., the 3' part of the conserved domain IV, is protected. The data suggest that a bend in the domain IV region, most likely at the central asymmetric internal loop, is an important element of the tertiary structure of 4.5S RNA. Hyperchromicity and lead cleavage data are consistent with the model as they reveal the unfolding of a higher-order structure between 30 and 40 degrees C. Protection by protein P48 occurs in this region of the RNA and, more strongly, in the 5' part of domain IV (nt 26-50, most strongly from 35 to 49). It is likely that P48 binds to the outside of the bent form of 4.5S RNA.     

Nucleotide sequences of the 4.5 S and 5 S ribosomal RNA genes from tobacco chloroplasts

Summary The DNA sequences of the 4.5 S and 5 S RNA genes from tobacco chloroplasts have been determined. The coding regions for the mature 4.5 S and 5 S RNAs were identified by sequencing these RNAs. The 4.5 S and 5 S RNA genes are composed of 103 and 121 base pairs respectively. These two genes are separated by the 256 base pair spacer. Several unique features in the spacer and in the region downstream from the 5 S coding region are discussed.     

Depletion of free 30S ribosomal subunits in Escherichia coli by expression of RNA containing Shine-Dalgarno-like sequences.

We have constructed synthetic coding sequences for the expression of poly(alpha,L-glutamic acid) (PLGA) as fusion proteins with dihydrofolate reductase (DHFR) in Escherichia coli. These PLGA coding sequences use both GAA and GAG codons for glutamic acid and contain sequence elements (5'-GAGGAGG-3') that resemble the consensus Shine-Dalgarno (SD) sequence found at translation initiation sites in bacterial mRNAs. An unusual feature of DHFR-PLGA expression is that accumulation of the protein is inversely related to the level of induction of its mRNA. Cellular protein synthesis was inhibited >95% by induction of constructs for either translatable or untranslatable PLGA RNAs. Induction of PLGA RNA resulted in the depletion of free 30S ribosomal subunits and the appearance of new complexes in the polyribosome region of the gradient. Unlike normal polyribosomes, these complexes were resistant to breakdown in the presence of puromycin. The novel complexes contained 16S rRNA, 23S rRNA, and PLGA RNA. We conclude that multiple noninitiator SD-like sequences in the PLGA RNA inhibit cellular protein synthesis by sequestering 30S small ribosomal subunits and 70S ribosomes in nonfunctional complexes on the PLGA mRNA.