Second-site suppression of RNase E essentiality by mutation of the deaD RNA helicase in Escherichia coli

Escherichia coli cells normally require RNase E activity to propagate and form colonies. Using random Tn10 insertion mutagenesis, we screened for second-site suppressor mutations that restore colony-forming ability (CFA) to E. coli cells lacking RNase E function and found mutations in three separate chromosomal loci that had this phenotype. Restoration of CFA by mutations in two of the genes identified was observed only in nutrient-poor medium, whereas the effects of mutation of the ATP-dependent RNA helicase DeaD were medium independent. Suppression of the rne mutant phenotype by inactivation of deaD was partial, as rne deaD doubly mutant bacteria had a greatly prolonged generation time and grew as filamentous chains in liquid medium. Moreover, we found that CFA restoration by deaD inactivation requires normal expression of the endogenous rng gene in doubly mutant rne deaD cells. Second-site suppression by deaD mutation was attributable specifically to ablation of the helicase activity of DeaD and was reversed by adventitious expression of RhlE or RNase R, both of which can unwind double-stranded RNA. Our results suggest a previously unsuspected role for RNA secondary structure as a determinant of RNase E essentiality.     

Temperature-hypersensitive sites of the flagellar switch component FliG in Salmonella enterica serovar typhimurium

Three flagellar proteins, FliG, FliM, and FliN (FliGMN), are the components of the C ring of the flagellar motor. The genes encoding these proteins are multifunctional; they show three different phenotypes (Fla(-), Mot(-), and Che(-)), depending on the sites and types of mutations. Some of the Mot(-) mutants previously characterized are found to be motile. Reexamination of all Mot(-) mutants in fliGMN genes so far studied revealed that many of them are actually temperature sensitive (TS); that is, they are motile at 20 degrees C but nonmotile at 37 degrees C. There were two types of TS mutants: one caused a loss of function that was not reversed by a return to the permissive temperature (rigid TS), and the other caused a loss that was reversed (hyper-TS). The rigid TS mutants showed an all-or-none phenotype; that is, once a structure was formed, the structure and function were stable against temperature shifts. All of fliM and fliN and most of the fliG TS mutants belong to this group. On the other hand, the hyper-TS mutants (three of the fliG mutants) showed a temporal swimming/stop phenotype, responding to temporal temperature shifts when the structure was formed at a permissive temperature. Those hyper-TS mutation sites are localized in the C-terminal domain of the FliG molecules at sites that are different from the previously proposed functional sites. We discuss a role for this new region of FliG in the torque generation of the flagellar motor.     

Intragenic suppressors of temperature-sensitive rne mutations lead to the dissociation of RNase E activity on mRNA and tRNA substrates in Escherichia coli

RNase E of Escherichia coli is an essential endoribonuclease that is involved in many aspects of RNA metabolism. Point mutations in the S1 RNA-binding domain of RNase E (rne-1 and rne-3071) lead to temperature-sensitive growth along with defects in 5S rRNA processing, mRNA decay and tRNA maturation. However, it is not clear whether RNase E acts similarly on all kinds of RNA substrates. Here we report the isolation and characterization of three independent intragenic second-site suppressors of the rne-1 and rne-3071 alleles that demonstrate for the first time the dissociation of the in vivo activity of RNase E on mRNA versus tRNA and rRNA substrates. Specifically, tRNA maturation and 9S rRNA processing were restored to wild-type levels in each of the three suppressor mutants (rne-1/172, rne-1/186 and rne-1/187), while mRNA decay and autoregulation of RNase E protein levels remained as defective as in the rne-1 single mutant. Each single amino acid substitution (Gly→Ala at amino acid 172; Phe → Cys at amino acid 186 and Arg → Leu at amino acid 187) mapped within the 5′ sensor region of the RNase E protein. Molecular models of RNase E suggest how suppression may occur.     

Characterization of a specific interaction between Escherichia coli thymidylate synthase and Escherichia coli thymidylate synthase mRNA.

Previous studies have shown that human TS mRNA translation is controlled by a negative autoregulatory mechanism. In this study, an RNA electrophoretic gel mobility shift assay confirmed a direct interaction between Escherichia coli (E.coli) TS protein and its own E.coli TS mRNA. Two cis-acting sequences in the E.coli TS mRNA protein-coding region were identified, with one site corresponding to nucleotides 207-460 and the second site corresponding to nucleotides 461-807. Each of these mRNA sequences bind TS with a relative affinity similar to that of the full-length E.coli TS mRNA sequence (IC50 = 1 nM). A third binding site was identified, corresponding to nucleotides 808-1015, although its relative affinity for TS (IC50 = 5.1 nM) was lower than that of the other two cis-acting elements. E.coli TS proteins with mutations in amino acids located within the nucleotide-binding region retained the ability to bind RNA while proteins with mutations at either the nucleotide active site cysteine (C146S) or at amino acids located within the folate-binding region were unable to bind TS mRNA. These studies suggest that the regions on E.coli TS defined by the folate-binding site and/or critical cysteine sulfhydryl groups may represent important RNA binding domains. Further evidence is presented which demonstrates that the direct interaction with TS results in in vitro repression of E.coli TS mRNA translation.     

Characterization of human immunodeficiency virus type 1 matrix revertants: effects on virus assembly, Gag processing, and Env incorporation into virions.

The matrix protein of human immunodeficiency virus type 1 (HIV-1) has been postulated to serve a variety of functions in the virus life cycle. Previously, we introduced a large number of mutations into the HIV-1 matrix and determined the effects on virus replication. These studies identified domains involved in virus assembly and release and envelope glycoprotein incorporation into virions. Here we describe the identification and characterization of viral revertants containing second-site changes in the matrix which compensate for the effects of four of the original mutations on matrix function. Specifically, mutations at matrix residues 4 and 6 severely impaired virus assembly and release; substitutions at residues 4 and 6 reversed the phenotype of the amino acid 4 change while second-site mutations at matrix positions 10, 69, and 97 partially or fully reversed the phenotype of the amino acid 6 substitution. A mutation at matrix residue 62 reversed the effect of a position 34 change which blocks envelope glycoprotein incorporation into virions, and substitutions at residues 27 and 51 reversed the phenotype of a position 86 mutation which redirects virus assembly to the cytoplasm. In addition to determining the effects of the compensatory changes in the context of the original mutations, we also introduced and analyzed the second-site changes alone in the context of the wild-type molecular clone. The data presented here define potential intermolecular and intramolecular interactions which occur in the matrix during the virus life cycle and have implications for our understanding of the relationship between matrix structure and function.     

The RNase E of Escherichia coli is a membrane-binding protein

RNase E is an essential endoribonuclease involved in RNA processing and mRNA degradation. The N-terminal half of the protein encompasses the catalytic domain; the C-terminal half is the scaffold for the assembly of the multienzyme RNA degradosome. Here we identify and characterize 'segment-A', an element in the beginning of the non-catalytic region of RNase E that is required for membrane binding. We demonstrate in vitro that an oligopeptide corresponding to segment-A has the propensity to form an amphipathic alpha-helix and that it avidly binds to protein-free phospholipid vesicles. We demonstrate in vitro and in vivo that disruption of segment-A in full-length RNase E abolishes membrane binding. Taken together, our results show that segment-A is necessary and sufficient for RNase E binding to membranes. Strains in which segment-A has been disrupted grow slowly. Since in vitro experiments show that phospholipid binding does not affect the ribonuclease activity of RNase E, the slow-growth phenotype might arise from a defect involving processes such as accessibility to substrates or interactions with other membrane-bound machinery. This is the first report demonstrating that RNase E is a membrane-binding protein and that its localization to the inner cytoplasmic membrane is important for normal cell growth.     

Identification and analysis of Escherichia coli ribonuclease E dominant-negative mutants

The Escherichia coli (E. coli) ribonuclease E protein (RNase E) is implicated in the degradation and processing of a large fraction of RNAs in the cell. To understand RNase E function in greater detail, we developed an efficient selection method for identifying nonfunctional RNase E mutants. A subset of the mutants was found to display a dominant-negative phenotype, interfering with wild-type RNase E function. Unexpectedly, each of these mutants contained a large truncation within the carboxy terminus of RNase E. In contrast, no point mutants that conferred a dominant-negative phenotype were found. We show that a representative dominant-negative mutant can form mixed multimers with RNase E and propose a model to explain how these mutants can block wild-type RNase E function in vivo.     

Suppressors of Cleavage-Site Mutations in the p62 Envelope Protein of Semliki Forest Virus Reveal Dynamics in Spike Structure and Function

The E2 spike glycoprotein of Semliki Forest virus is produced as a p62 precursor protein, which is cleaved by host proteases to its mature form, E2. Cleavage is not necessary for particle formation or release but is necessary for infectivity. Previous results had shown that phenotypic revertants of cleavage-deficient p62 mutants are generated, and here we show that these may contain second-site suppressor mutations in the vicinity of the cleavage site. These hot-spot sites were mutated to abolish the generation of such suppressor mutations; however, secondary mutations in another distant domain of the E2 protein appeared instead, all of which still caused cleavage-deficient mutations. Such mutants grew very poorly and were inefficient in virus entry and release. The mutated sites define domains of the spike protein which probably interact to regulate its structure and function. Because of their highly attenuated phenotype and the lower probability of reversion, the new mutations close to the cleavage site were used to make new helper vectors for packaging of recombinant RNA into infectious particles, thus increasing further the biosafety of the vector system based on the Semliki Forest virus replicon.     

RNase E polypeptides lacking a carboxyl-terminal half suppress a mukB mutation in Escherichia coli.

We have isolated suppressor mutants that suppress temperature-sensitive colony formation and anucleate cell production of a mukB mutation. A linkage group (smbB) of the suppressor mutations is located in the rne/ams/hmp gene encoding the processing endoribonuclease RNase E. All of the rne (smbB) mutants code for truncated RNase E polypeptides lacking a carboxyl-terminal half. The amount of MukB protein was higher in these rne mutants than that in the rne+ strain. These rne mutants grew nearly normally in the mukB+ genetic background. The copy number of plasmid pBR322 in these rne mutants was lower than that in the rne+ isogenic strain. The results suggest that these rne mutations increase the half-lives of mukB mRNA and RNAI of pBR322, the antisense RNA regulating ColE1-type plasmid replication. We have demonstrated that the wild-type RNase E protein bound to polynucleotide phosphorylase (PNPase) but a truncated RNase E polypeptide lacking the C-terminal half did not. We conclude that the C-terminal half of RNase E is not essential for viability but plays an important role for binding with PNPase. RNase E and PNPase of the multiprotein complex presumably cooperate for effective processing and turnover of specific substrates, such as mRNAs and other RNAs in vivo.     

Neighborhood properties are important determinants of temperature sensitive mutations

Temperature-sensitive (TS) mutants are powerful tools to study gene function in vivo. These mutants exhibit wild-type activity at permissive temperatures and reduced activity at restrictive temperatures. Although random mutagenesis can be used to generate TS mutants, the procedure is laborious and unfeasible in multicellular organisms. Further, the underlying molecular mechanisms of the TS phenotype are poorly understood. To elucidate TS mechanisms, we used a machine learning method-logistic regression-to investigate a large number of sequence and structure features. We developed and tested 133 features, describing properties of either the mutation site or the mutation site neighborhood. We defined three types of neighborhood using sequence distance, Euclidean distance, and topological distance. We discovered that neighborhood features outperformed mutation site features in predicting TS mutations. The most predictive features suggest that TS mutations tend to occur at buried and rigid residues, and are located at conserved protein domains. The environment of a buried residue often determines the overall structural stability of a protein, thus may lead to reversible activity change upon temperature switch. We developed TS prediction models based on logistic regression and the Lasso regularized procedure. Through a ten-fold cross-validation, we obtained the area under the curve of 0.91 for the model using both sequence and structure features. Testing on independent datasets suggested that the model predicted TS mutations with a 50% precision. In summary, our study elucidated the molecular basis of TS mutants and suggested the importance of neighborhood properties in determining TS mutations. We further developed models to predict TS mutations derived from single amino acid substitutions. In this way, TS mutants can be efficiently obtained through experimentally introducing the predicted mutations.     

Effects of point mutations in the major capsid protein of beet western yellows virus on capsid formation,virus accumulation,and aphid transmission

Point mutations were introduced into the major capsid protein (P3) of cloned infectious cDNA of the polerovirus beet western yellows virus (BWYV) by manipulation of cloned infectious cDNA. Seven mutations targeted sites on the S domain predicted to lie on the capsid surface. An eighth mutation eliminated two arginine residues in the R domain, which is thought to extend into the capsid interior. The effects of the mutations on virus capsid formation, virus accumulation in protoplasts and plants, and aphid transmission were tested. All of the mutants replicated in protoplasts. The S-domain mutant W166R failed to protect viral RNA from RNase attack, suggesting that this particular mutation interfered with stable capsid formation. The R-domain mutant R7A/R8A protected approximately 90% of the viral RNA strand from RNase, suggesting that lower positive-charge density in the mutant capsid interior interfered with stable packaging of the complete strand into virions. Neither of these mutants systemically infected plants. The six remaining mutants properly packaged viral RNA and could invade Nicotiana clevelandii systemically following agroinfection. Mutant Q121E/N122D was poorly transmitted by aphids, implicating one or both targeted residues in virus-vector interactions. Successful transmission of mutant D172N was accompanied either by reversion to the wild type or by appearance of a second-site mutation, N137D. This finding indicates that D172 is also important for transmission but that the D172N transmission defect can be compensated for by a "reverse" substitution at another site. The results have been used to evaluate possible structural models for the BWYV capsid.     

Genetic analysis of a synaptic calcium channel in Drosophila: intragenic modifiers of a temperature-sensitive paralytic mutant of cacophony

Our previous genetic analysis of synaptic mechanisms in Drosophila identified a temperature-sensitive paralytic mutant of the voltage-gated calcium channel alpha1 subunit gene, cacophony (cac). Electrophysiological studies in this mutant, designated cac(TS2), indicated cac encodes a primary calcium channel alpha1 subunit functioning in neurotransmitter release. To further examine the functions and interactions of cac-encoded calcium channels, a genetic screen was performed to isolate new mutations that modify the cac(TS2) paralytic phenotype. The screen recovered 10 mutations that enhance or suppress cac(TS2), including second-site mutations in cac (intragenic modifiers) as well as mutations mapping to other genes (extragenic modifiers). Here we report molecular characterization of three intragenic modifiers and examine the consequences of these mutations for temperature-sensitive behavior, synaptic function, and processing of cac pre-mRNAs. These mutations may further define the structural basis of calcium channel alpha1 subunit function in neurotransmitter release.     

A Streptomyces coelicolor Antibiotic Regulatory Gene, absB, Encodes an RNase III Homolog

Streptomyces coelicolor produces four genetically and structurally distinct antibiotics in a growth-phase-dependent manner. S. coelicolor mutants globally deficient in antibiotic production (Abs(-) phenotype) have previously been isolated, and some of these were found to define the absB locus. In this study, we isolated absB-complementing DNA and show that it encodes the S. coelicolor homolog of RNase III (rnc). Several lines of evidence indicate that the absB mutant global defect in antibiotic synthesis is due to a deficiency in RNase III. In marker exchange experiments, the S. coelicolor rnc gene rescued absB mutants, restoring antibiotic production. Sequencing the DNA of absB mutants confirmed that the absB mutations lay in the rnc open reading frame. Constructed disruptions of rnc in both S. coelicolor 1501 and Streptomyces lividans 1326 caused an Abs(-) phenotype. An absB mutation caused accumulation of 30S rRNA precursors, as had previously been reported for E. coli rnc mutants. The absB gene is widely conserved in streptomycetes. We speculate on why an RNase III deficiency could globally affect the synthesis of antibiotics.     

DNA base changes and RNA levels in N-acetoxy-2-acetylaminofluorene-induced dihydrofolate reductase mutants of Chinese hamster ovary cells

Formerly, we isolated a series of dihydrofolate reductase-deficient Chinese hamster ovary cell mutants that were induced by N-acetoxy-2-acetylaminofluorene. Deletions and complex gene rearrangements were detected in 28% of these mutants; 72% contained putative point mutations. In the present study, we have localized the putative point mutations in the 25,000 base dhfr gene by RNase heteroduplex mapping. Assignment of a position for each mutation was successful in 16 of 19 mutants studied. We cloned DNA fragments containing the mapped mutations from nine mutants into a bacteriophage lambda vector. In the case of 11 other mutants, DNA was amplified by the polymerase chain reaction procedure. Sequence analysis of cloned and amplified DNA confirmed the presence of point mutations. Most mutants (90%) carried base substitutions; the rest contained frameshift mutations. Of the point mutations, 75% were G.C to T.A transversions in either the dhfr coding sequence or at splice sites; transition G.C to A.T mutations were found in two mutants (10%). In one of these transition mutants, the base substitution occurred at the fifth base of the third intron. Of the frameshift mutations, one was a deletion of G.C pair and the other was an insertion of an A.T pair. Of the mapped mutants, 38% exhibited greatly reduced (approximately 10-fold) steady-state levels of dhfr mRNA. All eight sequenced mutants displaying this phenotype contained premature chain termination codons. Normal levels of dhfr mRNA were observed in five missense mutants and in five mutants carrying nonsense codons in the translated portion of exon VI. Taken together with the results of other mutagens at this locus, we conclude that the low dhfr mRNA phenotype is correlated with the presence of nonsense codons in exons II to V but not in the last exon of the dhfr gene.     

Mutations Altering the Predicted Secondary Structure of a Chloroplast 5′ Untranslated Region Affect Its Physical and Biochemical Properties as Well as Its Ability To Promote Translation of Reporter mRNAs Both in the Chlamydomonas reinhardtii Chloroplast and in Escherichia coli

Random mutations were generated in the sequence for the 5' untranslated region (5'UTR) of the Chlamydomonas reinhardtii chloroplast rps7 mRNA by PCR, the coding sequence for the mutant leaders fused upstream of the lacZ' reporter in pUC18, and transformed into Escherichia coli, and white colonies were selected. Twelve single base pair changes were found at different positions in the rps7 5'UTR in 207 white colonies examined. Seven of the 12 mutant leaders allowed accumulation of abundant lacZ' message. These mutant rps7 leaders were ligated into an aadA expression cassette and transformed into the chloroplast of C. reinhardtii and into E. coli. In vivo spectinomycin-resistant growth rates and in vitro aminoglycoside adenyltransferase enzyme activity varied considerably between different mutants but were remarkably similar for a given mutant expressed in the Chlamydomonas chloroplast and in E. coli. The variable effect of the mutants on aadA reporter expression and their complete abolition of lacZ' reporter expression in E. coli suggests differences in the interaction between the 5'UTR of rps7 and aadA or lacZ' coding regions. Several rps7 5'UTR mutations affected the predicted folding pattern of the 5'UTR by weakening the stability of stem structures. Site-directed secondary mutations generated to restore these structures in the second stem suppressed the loss of reporter activity caused by the original mutations. Additional site-directed mutations that were predicted to further strengthen (A-U-->G-C) or weaken (G-C-->A-U) the second stem of the rps7 leader both resulted in reduced reporter expression. This genetic evidence combined with differences between mutant and wild-type UV melting profiles and RNase T1 protection gel shifts further indicate that the predicted wild-type folding pattern in the 5'UTR is likely to play an essential role in translation initiation.     

Dark-induced mRNA instability involves RNase E/G-type endoribonuclease cleavage at the AU-box and SD sequences in cyanobacteria

Light-responsive gene expression is crucial to photosynthesizing organisms. Here, we studied functions of cis-elements (AU-box and SD sequences) and a trans-acting factor (ribonuclease, RNase) in light-responsive expression in cyanobacteria. The results indicated that AU-rich nucleotides with an AU-box, UAAAUAAA, just upstream from an SD confer instability on the mRNA under darkness. An RNase E/G homologue, Slr1129, of the cyanobacterium Synechocystis sp. strain PCC 6803 was purified and confirmed capable of endoribonucleolytic cleavage at the AU- (or AG)-rich sequences in vitro. The cleavage depends on the primary target sequence and secondary structure of the mRNA. Complementation tests using Escherichia coli rne/rng mutants showed that Slr1129 fulfilled the functions of both the RNase E and RNase G. An analysis of systematic mutations in the AU-box and SD sequences showed that the cis-elements also affect significantly mRNA stability in light-responsive genes. These results strongly suggested that dark-induced mRNA instability involves RNase E/G-type cleavage at the AU-box and SD sequences in cyanobacteria. The mechanical impact and a possible common mechanism with RNases for light-responsive gene expression are discussed. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.