Phylogenetic and biochemical evidence for a secondary structure model of a small cytoplasmic RNA from Bacilli.

Small cytoplasmic RNA (scRNA; 271 nucleotides) is an abundant, stable RNA identified in the Gram-positive eubacterium Bacillus subtilis. Several findings suggest an important role of scRNA in protein biosynthesis: it shares structural and biochemical features with the Escherichia coli 4.5S RNA (114 nucleotides), a molecule known to be involved in this process, and it can complement the essential function of 4.5S RNA in vivo. The common apical hairpin motif of scRNA and 4.5S RNA also exists in eukaryotic 7SL RNA, the RNA component of the signal recognition particle. To elucidate the higher-order structure of scRNA, we have combined a phylogenetic approach with a biochemical one. The sequence of scRNA from a thermophilic relative of B. subtilis, Bacillus stearothermophilus, was determined and compared with the B. subtilis scRNA. In addition, the solution structure of B. stearothermophilus scRNA was probed with single- and double-strand-specific nucleases. Both types of analysis support a secondary structure model for scRNA that strongly resembles 4.5S RNA and respective parts of 7SL RNA. The results provide further evidence for the suggestion of a functional relationship between these RNAs.     

Bacillus subtilis histone-like protein, HBsu, is an integral component of a SRP-like particle that can bind the Alu domain of small cytoplasmic RNA

Small cytoplasmic RNA (scRNA) is metabolically stable and abundant in Bacillus subtilis cells. Consisting of 271 nucleotides, it is structurally homologous to mammalian signal recognition particle RNA. In contrast to 4.5 S RNA of Escherichia coli, B. subtilis scRNA contains an Alu domain in addition to the evolutionarily conserved S domain. In this study, we show that a 10-kDa protein in B. subtilis cell extracts has scRNA binding activity at the Alu domain. The in vitro binding selectivity of the 10-kDa protein shows that it recognizes the higher structure of the Alu domain of scRNA caused by five consecutive complementary sequences in the two loops. Purification and subsequent analyses demonstrated that the 10-kDa protein is HBsu, which was originally identified as a member of the histone-like protein family. By constructing a HBsu-deficient B. subtilis mutant, we showed that HBsu is essential for normal growth. Immunoprecipitating cell lysates using anti-HBsu antibody yielded scRNA. Moreover, the co-precipitation of HBsu with (His)6-tagged Ffh depended on the presence of scRNA, suggesting that HBsu, Ffh, and scRNA make a ternary complex and that scRNA serves as a functional unit for binding. These results demonstrated that HBsu is the third component of a signal recognition particle-like particle in B. subtilis that can bind the Alu domain of scRNA.     

Structural requirements of Bacillus subtilis small cytoplasmic RNA for cell growth, sporulation, and extracellular enzyme production.

Bacillus subtilis small cytoplasmic RNA (scRNA; 271 nucleotides) is a member of the signal recognition particle (SRP) RNA family, which has evolutionarily conserved primary and secondary structures. The scRNA consists of three domains corresponding to domains I, II, and IV of human SRP 7S RNA. To identify the structural determinants required for its function, we constructed mutant scRNAs in which individual domains or conserved nucleotides were deleted, and their importance was assayed in vivo. The results demonstrated that domain IV of scRNA is necessary to maintain cell viability. On the other hand, domains I and II were not essential for vegetative growth but were preferentially required for the RNA to achieve its active structure, and assembled ribonucleoprotein between Ffh and scRNA is required for sporulation to proceed. This view is highly consistent with the fact that the presence of domains I and II is restricted to sporeforming B. subtilis scRNA among eubacterial SRP RNA-like RNAs.     

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.     

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.     

Molecular cloning and phylogenetic analysis of the small cytoplasmic RNA from Listeria monocytogenes

A molecular cloning strategy has been designed to isolate the gene that encodes the small cytoplasmic RNA (scRNA) component of bacterial signal recognition particles. Using this strategy a putative Listeria monocytogenes scRNA lambda gt11 recombinant clone was isolated. A previously described complementation assay developed to genetically select functional homologues of 4.5S RNA and scRNA of bacteria confirmed that the lambda gt11 recombinant clone isolated encoded for the scRNA from L. monocytogenes. A secondary structure for this scRNA is proposed and a phylogenetic comparison of the 276 base L. monocytogenes scRNA with previously characterised Gram-positive bacterial scRNAs is also presented.     

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.     

Evolutionary conserved nucleotides within the E.coli 4.5S RNA are required for association with P48 in vitro and for optimal function in vivo.

E.coli 4.5S RNA is homologous to domain IV of eukaryotic SPR7S RNA, the RNA component of the signal recognition particle. The 4.5S RNA is associated in vivo with a 48kD protein (P48), which is homologous to a protein component of the signal recognition particle, SRP54. In addition to secondary structural features, a number of nucleotides are conserved between the 4.5S RNA and domain IV of all other characterised SRP-like RNAs from eubacteria, arachaebacteria and eukaryotes. This domain consists of an extended stem-loop structure; conserved nucleotides lie within the terminal loop and within single-stranded regions bulged from the stem immediately preceding the loop. This conserved region is a candidate for the SRP54/P48 binding site. To determine the functional importance of this region within the 4.5S RNA, mutations were introduced into the 4.5S RNA coding sequence. Mutated alleles were tested for their function in vivo and for the ability of the corresponding RNAs to bind P48 in vitro. Single point mutations in conserved nucleotides within the terminal tetranucleotide loop do not affect P48 binding in vitro and produce only slight growth defects. This suggests that the sequence of the loop may be important for the structure of the molecule rather than for specific interactions with P48. On the other hand, nucleotides within the single-stranded regions bulged from the stem were found to be important both for the binding of P48 to the RNA and for optimal function of the RNA in vivo.     

Conformation of 4.5S RNA in the signal recognition particle and on the 30S ribosomal subunit

The signal recognition particle (SRP) from Escherichia coli consists of 4.5S RNA and protein Ffh. It is essential for targeting ribosomes that are translating integral membrane proteins to the translocation pore in the plasma membrane. Independently of Ffh, 4.5S RNA also interacts with elongation factor G (EF-G) and the 30S ribosomal subunit. Here we use a cross-linking approach to probe the conformation of 4.5S RNA in SRP and in the complex with the 30S ribosomal subunit and to map the binding site. The UV-activatable cross-linker p-azidophenacyl bromide (AzP) was attached to positions 1, 21, and 54 of wild-type or modified 4.5S RNA. In SRP, cross-links to Ffh were formed from AzP in all three positions in 4.5S RNA, indicating a strongly bent conformation in which the 5' end (position 1) and the tetraloop region (including position 54) of the molecule are close to one another and to Ffh. In ribosomal complexes of 4.5S RNA, AzP in both positions 1 and 54 formed cross-links to the 30S ribosomal subunit, independently of the presence of Ffh. The major cross-linking target on the ribosome was protein S7; minor cross-links were formed to S2, S18, and S21. There were no cross-links from 4.5S RNA to the 50S subunit, where the primary binding site of SRP is located close to the peptide exit. The functional role of 4.5S RNA binding to the 30S subunit is unclear, as the RNA had no effect on translation or tRNA translocation on the ribosome.     

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.     

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.     

Domain rearrangement of SRP protein Ffh upon binding 4.5S RNA and the SRP receptor FtsY

The signal recognition particle (SRP) mediates membrane targeting of translating ribosomes displaying a signal-anchor sequence. In Escherichia coli, SRP consists of 4.5S RNA and a protein, Ffh, that recognizes the signal peptide emerging from the ribosome and the SRP receptor at the membrane, FtsY. In the present work, we studied the interactions between the NG and M domains in Ffh and their rearrangements upon complex formation with 4.5S RNA and/or FtsY. In free Ffh, the NG and M domains are facing one another in an orientation that allows cross-linking between positions 231 in the G domain and 377 in the M domain. There are binding interactions between the two domains, as the isolated domains form a strong complex. The interdomain contacts are disrupted upon binding of Ffh to 4.5S RNA, consuming a part of the total binding energy of 4.5S RNA-Ffh association that is roughly equivalent to the free energy of domain binding to each other. In the SRP particle, the NG domain binds to 4.5S RNA in a region adjacent to the binding site of the M domain. Ffh binding to FtsY also requires a reorientation of NG and M domains. These results suggest that in free Ffh, the binding sites for 4.5S RNA and FtsY are occluded by strong domain-domain interactions which must be disrupted for the formation of SRP or the Ffh-FtsY complex.     

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.     

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.     

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.     

Regulation of 6S RNA by pRNA synthesis is required for efficient recovery from stationary phase in E. coli and B. subtilis

6S RNAs function through interaction with housekeeping forms of RNA polymerase holoenzyme (Eσ(70) in Escherichia coli, Eσ(A) in Bacillus subtilis). Escherichia coli 6S RNA accumulates to high levels during stationary phase, and has been shown to be released from Eσ(70) during exit from stationary phase by a process in which 6S RNA serves as a template for Eσ(70) to generate product RNAs (pRNAs). Here, we demonstrate that not only does pRNA synthesis occur, but it is an important mechanism for regulation of 6S RNA function that is required for cells to exit stationary phase efficiently in both E. coli and B. subtilis. Bacillus subtilis has two 6S RNAs, 6S-1 and 6S-2. Intriguingly, 6S-2 RNA does not direct pRNA synthesis under physiological conditions and its non-release from Eσ(A) prevents efficient outgrowth in cells lacking 6S-1 RNA. The behavioral differences in the two B. subtilis RNAs clearly demonstrate that they act independently, revealing a higher than anticipated diversity in 6S RNA function globally. Overexpression of a pRNA-synthesis-defective 6S RNA in E. coli leads to decreased cell viability, suggesting pRNA synthesis-mediated regulation of 6S RNA function is important at other times of growth as well.     

Depletion of Bacillus subtilis histone-like protein, HBsu, causes defective protein translocation and induces upregulation of small cytoplasmic RNA

Small cytoplasmic RNA (scRNA) is a metabolically stable homologue of mammalian SRP RNA that contains an Alu-like domain. The Bacillus subtilis histone-like protein HBsu can bind this domain. We demonstrate here that repressing the level of HBsu results in slow growth and the accumulation of precursor of beta-lactamase fusion proteins having the signal sequence of alkaline protease, penicillin binding protein 5* (PBP5*) or CGTase. The degree of the translocation defect varied among the various signal sequences tested. A pulse-chase experiment showed that processing the alpha-amylase signal sequence is significantly inhibited in HBsu-depleted cells. Northern blot analysis indicated that repressing the HBsu gene induces scRNA upregulation, indicating that the defective translocation of presecretory proteins is not due to a reduced scRNA level. The data presented here suggest that HBsu plays a pivotal role in SRP function rather than simply stabilizing the other SRP components such as scRNA.