Conserved but nonessential interaction of SRP RNA with translation factor EF-G

4.5S RNA is essential for viability of Escherichia coli, and forms a key component of the signal recognition particle (SRP), a ubiquitous ribonucleoprotein complex responsible for cotranslational targeting of secretory proteins. 4.5S RNA also binds independently to elongation factor G (EF-G), a five-domain GTPase that catalyzes the translocation step during protein biosynthesis on the ribosome. Point mutations in EF-G suppress deleterious effects of 4.5S RNA depletion, as do mutations in the EF-G binding site within ribosomal RNA, suggesting that 4.5S RNA might play a critical role in ribosome function in addition to its role in SRP. Here we show that 4.5S RNA and EF-G form a phylogenetically conserved, low-affinity but highly specific complex involving sequence elements required for 4.5S binding to its cognate SRP protein, Ffh. Mutational analysis indicates that the same molecular structure of 4.5S RNA is recognized in each case. Surprisingly, however, the suppressor mutant forms of EF-G bind very weakly or undetectably to 4.5S RNA, implying that cells can survive 4.5S RNA depletion by decreasing the affinity between 4.5S RNA and the translational machinery. These data suggest that SRP function is the essential role of 4.5S RNA in bacteria.     

E. coli 4.5S RNA is part of a ribonucleoprotein particle that has properties related to signal recognition particle

E. coli 4.5S RNA and P48 have been shown to be homologous to SRP7S RNA and SRP54, respectively. Here we report that expression of human SRP7S in E. coli can suppress the lethality caused by depletion of 4.5S RNA. In E. coli, both RNAs are associated with P48. In vitro, both E. coli P48 and SRP54 specifically bind to 4.5S RNA. Strains depleted of 4.5S RNA strongly accumulate pre-beta-lactamase and fail to accumulate maltose binding protein. These effects commence well before any growth defect is observed and are suppressed by expression of human SRP7S. Strains overproducing P48 also accumulate pre-beta-lactamase. 4.5S RNA and P48 are components of a ribonucleoprotein particle that we propose to be required for the secretion of some proteins.     

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.     

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.     

Role for both DNA and RNA in GTP hydrolysis by the Neisseria gonorrhoeae signal recognition particle receptor

The prokaryotic signal recognition particle (SRP) targeting system is a complex of two proteins, FtsY and Ffh, and a 4.5S RNA that targets a subset of proteins to the cytoplasmic membrane cotranslationally. We previously showed that Neisseria gonorrhoeae PilA is the gonococcal FtsY homolog. In this work, we isolated the other two components of the gonococcal SRP, Ffh and 4.5S RNA, and characterized the interactions among the three SRP components by using gel retardation and nitrocellulose filter-binding assays and enzymatic analyses of the two proteins. In the current model of prokaryotic SRP function, based on studies of the Escherichia coli and mammalian systems, Ffh binds to 4.5S RNA and the Ffh-4.5S RNA complex binds to the signal sequence of nascent peptides and then docks with FtsY at the membrane. GTP is hydrolyzed by both proteins synergistically, and the nascent peptide is transferred to the translocon. We present evidence that the in vitro properties of the gonococcal SRP differ from those of previously described systems. GTP hydrolysis by PilA, but not that by Ffh, was stimulated by 4.5S RNA, suggesting a direct interaction between PilA and 4.5S RNA that has not been reported in other systems. This interaction was confirmed by gel retardation analyses in which PilA and Ffh, both alone and together, bound to 4.5S RNA. An additional novel finding was that P(pilE) DNA, previously shown by us to bind PilA in vitro, also stimulates PilA GTP hydrolysis. On the basis of these data, we hypothesize that DNA may play a role in targeting proteins via the SRP.     

Ribonucleic Acid and Protein Synthesis in a Mutant of Bacillus subtilis Defective in Potassium Retention

A mutant of Bacillus subtilis 168 (strain 168 KL), which had lost its normal capacity to accumulate K(+), was used to explore the interrelationship between protein and ribonucleic acid (RNA) synthesis. In contrast to the wild type, the growth rate of strain 168 KL was markedly dependent on the K(+) concentration in the medium. K(+) uptake in the mutant strain was identical to that in the parent, but the mutant was unable to retain and accumulate K(+). Protein synthesis was markedly dependent on the K(+) concentration in the medium, whereas RNA synthesis was relatively unaffected by changes in the level of K(+). Most of the RNA synthesized during K(+) depletion was ribosomal RNA; it appeared in crude extracts in the form of ribonucleoproteins particles with sedimentation values between 4S and 30S. These particles were converted into mature ribosomes when growth was allowed to resume by the addition of K(+). Simultaneous synthesis of RNA and protein was necessary for the quantitative conversion of the ribonucleoprotein particles into ribosomes. During recovery from K(+) depletion, ribosomal protein was synthesized in preference to the other proteins of the cell.     

Role of SRP RNA in the GTPase cycles of Ffh and FtsY.

The bacterial homologues of the signal recognition particle (SRP) and its receptor, the Ffh*4.5S RNA ribonucleoprotein complex and the FtsY protein, respectively, form a unique complex in which both Ffh and FtsY act as GTPase activating proteins for one another, resulting in the mutual stimulation of GTP hydrolysis by both proteins. Previous work showed that 4.5S RNA enhances the GTPase activity in the presence of both Ffh and FtsY, but it was not clear how this was accomplished. In this work, kinetic and thermodynamic analyses of the GTPase reactions of Ffh and FtsY have provided insights into the role of 4.5S RNA in the GTPase cycles of Ffh and FtsY. We found that 4.5S RNA accelerates the association between Ffh and FtsY 400-fold in their GTP-bound form, analogous to its 200-fold catalytic effect on Ffh*FtsY association previously observed with the GppNHp-bound form [Peluso, P., et al. (2000) Science 288, 1640-1643]. Further, Ffh-FtsY association is rate-limiting for the observed GTPase reaction with subsaturating Ffh and FtsY, thereby accounting for the apparent stimulatory effect of 4.5S RNA on the GTPase activity observed previously. An additional step, GTP hydrolysis from the Ffh*FtsY complex, is also moderately facilitated by 4.5S RNA. These results suggest that 4.5S RNA modulates the conformation of the Ffh*FtsY complex and may, in turn, regulate its GTPase activity during the SRP functional cycle.     

Mammalian and Escherichia coli signal recognition particles

Recent evidence from both biochemical and genetic studies indicates that protein targeting to the pro-karyotic cytoplasmic membrane and the eukaryotic endoplasmic reticulum membrane may have more in common than previously thought. A ribonucleo-protein particle was identified in Escherichia coli that consists of at least one protein (P48 or Ffh) and one RNA molecule (4.5S RNA), both of which exhibit strong sequence similarity with constituents of the mammalian signal recognition particle (SRP). Like the mammalian SRP, the E. coli SRP binds specifically to the signal sequence of presecretory proteins. Depletion of either P48 or 4.5S RNA affects translation and results in the accumulation of precursors of several secreted proteins. This review discusses these recent studies and speculates on the position of the SRP in the complex network of protein interactions involved in translation and membrane targeting in E. coli.     

Synthesis of secretory proteins in developing mouse yolk sac

Synthesis of secretory proteins in the developing mouse visceral yolk sac was studied. Newly synthesized proteins were labeled with [³⁵S]methionine and characterized by two-dimensional gel electrophoresis. A large increase in the relative rate of synthesis of a small number of proteins occurred between Days 9.5 and 15.5 of development. These proteins were the predominant proteins synthesized and secreted by the yolk sac throughout this period of gestation. Two of these proteins were identified as α-fetoprotein and transferrin by specific immunoprecipitation. α-Fetoprotein synthesis increased from about 3% of the total protein synthesis at Day 9.5 to about 26% at Day 15.5 after which it declined slightly. The relative rate of transferrin synthesis had a similar developmental pattern, reaching the highest level (5%) at Day 15.5, but declined more rapidly than α-fetoprotein synthesis. Quantitatively, these two proteins represented about 60% of the total secreted protein. Gestational changes in the content of α-fetoprotein messenger RNA were determined by hybridization analysis using α-fetoprotein complementary DNA probe. The percentage of α-fetoprotein messenger RNA in total yolk sac RNA increased about ninefold from Day 9.5 to Day 14.5. This increase correlated well with the increase in the relative rate of α-fetoprotein synthesis during the identical period. This study suggests that after Day 9.5 the yolk sac is completing a differentiation process which is characterized by the preferential expression of a small group of secretory protein genes.     

Dexamethasone effect on the growth rate, the plasminogen activator, and the MMTV RNA dependent DNA polymerase in a mouse mammary tumor cell line

We have compared the effect of dexamethasone on the growth rate and the accumulation of two secreted proteins of an established cell line of mouse mammary carcinoma (GR). Whereas overall protein synthesis is not affected; the cell growth rate and both intracellular and secreted plasminogen activator are inhibited by dexamethasone treatment. In contrast the viral RNA dependent DNA polymerase secreted with viral particles is strongly stimulated by physiological concentrations of dexamethasone. These results, discussed with others, suggest that glucocorticoids regulate specifically but in opposite ways the synthesis of two secreted proteins.     

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.     

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.     

Unbalanced rRNA gene dosage and its effects on rRNA and ribosomal-protein synthesis.

The synthesis of rRNA was unbalanced by the introduction of plasmids containing rRNA operons with large internal deletions. Significant unbalanced synthesis was achieved only when the deletions affected both 16S and 23S RNA genes or when the deletions affected the 23S RNA gene alone. Although large imbalances in rRNA synthesis resulted from deletions affecting 16S and 23S RNA genes or only 23S RNA genes, excess 16S RNA and defective rRNA species were rapidly degraded. Large imbalances in the synthesis of regions of rRNA did not result in significantly unbalanced synthesis of ribosomal proteins. It therefore is probable that excess intact 16S RNA is degraded because ribosomal proteins are not available for packaging the RNA into ribosomes. Defective RNA species also may be degraded for this reason or because proper ribosome assembly is prevented by the defects in RNA structure. We propose two possible explanations for the finding that unbalanced overproduction of binding sites for feedback ribosomal protein does not result in significant unbalanced translational feedback depression of ribosomal protein mRNAs.     

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.     

Mouse nucleolin binds to 4.5S RNAh, a small noncoding RNA

4.5S RNAh is a rodent-specific small noncoding RNA that exhibits extensive homology to the B1 short interspersed element. Although 4.5S RNAh is known to associate with cellular poly(A)-terminated RNAs and retroviral genomic RNAs, its function remains unclear. In this study, we analyzed 4.5S RNAh-binding proteins in mouse nuclear extracts using gel mobility shift and RNA-protein UV cross-linking assays. We found that at least nine distinct polypeptides (p170, p110, p93, p70, p48, p40, p34, p20, and p16.5) specifically interacted with 4.5S RNAhin vitro. Using anti-La antibody, p48 was identified as mouse La protein. To identify the other 4.5S RNAh-binding proteins, we performed expression cloning from a mouse cDNA library and obtained cDNA clones derived from nucleolin mRNA. We identified p110 as nucleolin using nucleolin-specific antibodies. UV cross-linking analysis using various deletion mutants of nucleolin indicated that the third of four tandem RNA recognition motifs is a major determinant for 4.5S RNAh recognition. Immunoprecipitation of nucleolin from the subcellular fractions of mouse cell extracts revealed that a portion of the endogenous 4.5S RNAh was associated with nucleolin and that this complex was located in both the nucleoplasm and nucleolus.     

RNA synthesis in Escherichia coli during K + -depletion

During K⁺ depletion of a mutant of $\text{Escherichia}$$\text{coli}$ which cannot concentrate this cation, protein synthesis is inhibited but RNA formation continues. The RNA produced during K⁺ depletion was analyzed by gel electrophoresis. It was found that 4S, 5S and 23S RNA were synthesized by K⁺-depleted cells whether uninfected or infected with phage T4. In addition, an RNA species moving close to 16S (presumably 17S) and material of about 6–10S were made during K⁺ depletion. These species of RNA were not evident in growing cells. Methylation of RNA is severely inhibited during K⁺ depletion.     

Methionine sulfoxide reductases protect Ffh from oxidative damages in Escherichia coli

In proteins, methionine residues are primary targets for oxidation. Methionine oxidation is reversed by methionine sulfoxide reductases A and B, a class of highly conserved enzymes. Ffh protein, a component of the ubiquitous signal recognition particle, contains a methionine-rich domain, interacting with a small 4.5S RNA. In vitro analyses reported here show that: (i) oxidized Ffh is unable to bind 4.5S RNA, (ii) oxidized Ffh contains methionine sulfoxide residues, (iii) oxidized Ffh is a substrate for MsrA and MsrB enzymes; and (iv) MsrA/B repairing activities allow oxidized Ffh to recover 4.5S RNA-binding abilities. In vivo analyses reveal that: (i) Ffh synthesized in the msrA msrB mutant contains methionine sulfoxide residues and is unstable, (ii) msrA msrB mutant requires high levels of Ffh synthesis for growth and (iii) msrA msrB mutation leads to defects in Ffh-dependent targeting of MalF. We conclude that MsrA and MsrB are required to repair Ffh oxidized by reactive oxygen species produced by aerobic metabolism, establishing an as-yet undescribed link between protein targeting and oxidation.