A conditionally lethal mutation of Escherichia coli affecting the gene coding for ribosomal protein S2 (rpsB).

Localized P1 mutagenesis has been used to isolate conditionally lethal mutations in the four-minute region of the Escherichia coli genome. One such mutation, ts25, has been mapped at about 3.7 minutes between the popC and dapD genes. This mutation leads to thermosensitivity of growth and impaired in vivo assembly of 30 S ribosomal subunits at 42 °C. The strain carrying the mutation has an altered S2 ribosomal protein as judged by (1) its inability to maintain stable complex with the ribosome under mild washing conditions and (2) its altered electrophoretic mobility.Spontaneous reversion to temperature independence can restore both the normal assembly in vivo of 30 S ribosomal subunits at 42 °C and the normal electrophoretic behaviour of the S2 ribosomal protein in vitro.We conclude therefore that the ts25 mutation affects the structural gene for ribosomal protein S2 (rpsB).     

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.     

A new mutation affecting ribosomal RNA synthesis in Escherichia coli.

A temperature-sensitive mutant of Escherichia coli is described. At the nonpermissive temperature there is a 12-fold reduction in the rate of rRNA synthesis, while tRNA and mRNA syntheses are affected to only a slight extent. Both protein and DNA syntheses also continue at nearly the normal rate. The mutation appears to affect the synthesis of 16S and 23S rRNA equally and has no detectable affect on rRNA maturation. The temperature-sensitive lesion appears to be caused by a single point mutation lying between minutes 21 and 27. It is suggested that this mutation seems to define a new genetic locus involved in the regulation of rRNA synthesis.     

Genetic analysis of a mutation affecting ribosomal protein S1 in Escherichia coli.

Summary Ribosomal protein S1 from a newly isolated Escherichia coli mutant has a molecular weight of about 54,000 which is smaller than the wild type S1 (M.W. 65,000). The isoelectric points of the smaller and the wild type S1 species are similar in the gel electrophoresis system of O'Farrell (1975). Genetic analyses by Hfr conjugation and P1 phage transduction indicate that the mutation affecting S1 (rpsA) is located close to the serC gene [20 min on the E. coli genetic map of Bachmann et al. (1976)], with a co-transduction frequency of 61%. The most probable gene order is serC-rpsA-cmlB.     

CsdA, a cold-shock RNA helicase from Escherichia coli, is involved in the biogenesis of 50S ribosomal subunit

CsdA, a DEAD-box protein from Escherichia coli, has been proposed to participate in a variety of processes, such as translation initiation, gene regulation after cold-shock, mRNA decay and biogenesis of the small ribosomal subunit. Whether the protein really plays a direct role in these multiple processes is however, not clear. Here, we show that CsdA is involved in the biogenesis of the large rather than the small ribosomal subunit. Deletion of the csdA gene leads to a deficit in free 50S subunits at low temperatures and to the accumulation of a new particle sedimenting around 40S. Analysis of the RNA and protein contents of this particle indicates that it corresponds to a mis-assembled large subunit. Sucrose gradient fractionation shows that in wild-type cells CsdA associates mainly with a pre50S particle. Presumably the RNA helicase activity of CsdA permits a structural rearrangement during 50S biogenesis at low temperature. We showed previously that SrmB, another DEAD-box RNA helicase, is also involved in 50S assembly in E.coli. Our results suggest that CsdA is required at a later step than SrmB. However, over-expression of CsdA corrects the ribosome defect of the srmB-deleted strain, indicating that some functional overlap exists between the two proteins.     

The binding site for ribosomal protein L2 within 23S ribosomal RNA of Escherichia coli.

Ribosomal protein L2 from Escherichia coli binds to and protects from nuclease digestion a substantial portion of 'domain IV' of 23S rRNA. In particular, oligonucleotides derived from the sequence 1757-1935 were isolated and shown to rebind specifically to protein L2 in vitro. Other L2-protected oligonucleotides, also derived from domain IV (i.e. from residues 1955-2010) did not rebind to protein L2 in vitro nor did others derived from domain I. Given that protein L2 is widely believed to be located in the peptidyl transferase centre of the 50S ribosomal subunit, these data suggest that domain IV of 23S rRNA is also present in that active site of the ribosomal enzyme.     

Genetic analysis of a mutation affecting ribosomal protein S1 in Escherichia coli

Molecular Genetics and Genomics - Ribosomal protein S1 from a newly isolated Escherichia coli mutant has a molecular weight of about 54,000 which is smaller than the wild type S1 (M.W. 65,000). The...     

An amber mutation in the gene encoding the beta'' subunit of Escherichia coli RNA polymerase.

An Escherichia coli strain carrying an amber mutation (UAG) in rpoC, the gene encoding the beta prime subunit of RNA polymerase, was isolated after mutagenesis with nitrosoguanidine. The mutation was moved into an unmutagenized strain carrying the supD43,74 allele, which encodes a temperature-sensitive su1 amber suppressor, and sue alleles, which enhance the efficiency of the suppressor. In this background, beta prime is not synthesized at high temperature. Suppression of the mutation by the non-temperature-sensitive amber suppressor su1+ yields a protein which is functional at all temperatures examined (30, 37, and 42 degrees C).     

The chloroplast gene encoding ribosomal protein S4 in Chlamydomonas reinhardtii spans an inverted repeat — unique sequence junction and can be mutated to suppress a streptomycin dependence mutation in ribosomal protein S12

The ribosomal protein gene rps4 was cloned and sequenced from the chloroplast genome of Chlamydomonas reinhardtii. The N-terminal 213 amino acid residues of the S4 protein are encoded in the single-copy region (SCR) of the genome, while the C-terminal 44 amino acid residues are encoded in the inverted repeat (IR). The deduced 257 amino acid sequence of C. reinhardtii S4 is considerably longer (by 51–59 residues) than S4 proteins of other photosynthetic species and Escherichia coli, due to the presence of two internal insertions and a C-terminal extension. A short conserved C-terminal motif found in all other S4 proteins examined is missing from the C. reinhardtii protein. In E. coli, mutations in the S4 protein suppress the streptomycin-dependent (sd) phenotype of mutations in the S12 protein. Because we have been unable to identify similar S4 mutations among suppressors of an sd mutation in C. reinhardtii S12 obtained using UV mutagenesis, we made site-directed mutations [Arg₆₈ (CGT) to Len (CTG and CTT)] in the wild-type rps4 gene equivalent to an E. coli Gln₅₃ to Len ribosomal ambiguity mutation (ram), which suppresses the sd phenotype and decreases translational accuracy. These mutants were tested for their ability to transform the sd S 12 mutation of C. reinhardtii to streptomycin independence. The streptomycin-independent isolates obtained by biolistic transformation all possessed the original sd mutation in rps12, but none had the expected donor Leu₆₈ mutations in rps4. Instead, six of 15 contained a Gln₇₃ (CAA) to Pro (CCA) mutation five amino acids downstream from the predicted mutant codon, irrespective of rps4 donor DNA. Two others contained six- and ten-amino acid, in-frame insertions at S4 positions 90 and 92 that appear to have been induced by the biolistic process itself. Eight streptomycin-independent isolates analyzed had wild-type rps4 genes and may possess mutations identical to previously isolated suppressors of sd that define at least two additional chloroplast loci. Cloned rps4 genes from streptomycin-independent isolates containing the Gln₇₃ to Pro mutation and the 6-amino acid insertion in r-protein S4 transform the sd strain to streptomycin independence.     

Escherichia coli ribosomal protein S1 recognizes two sites in bacteriophage Qbeta RNA.

As a component of bacteriophage Qbeta replicase, S1 is required both for initiation of Qbeta minus strand RNA synthesis and for translational repression, which has been traced to the ability of the enzyme to bind to an internal site in the Qbeta RNA molecule. Previously, Senear and Steitz (Senear, A. W., and Steitz, J. A. (1976) J. Biol. Chem. 251, 1902-1912) found that isolated S1 protein binds specifically to an oligonucleotide spanning residues -38 to -63 from the 3' terminus of Qbeta RNA. Here we report that S1 also interacts strongly with a second oligonucleotide in Qbeta RNA, which is derived from the region recognized by replicase just 5' to the Qbeta coat protein cistron. Both sequences exhibit pyrimidine-rich regions.     

A new ribosomal mutation which affects the two ribosomal subunits in Escherichia coli.

Summary The nif cistrons indentified by complementation analysis in the preceding paper (Dixon et al., 1977) were mapped with respect to hisD and to each other by Pl cotransduction and three-factor reciprocal crosses. The order obtained was hisD nifB nifA (nifL) nifF nifE nifK nifD nifH. Analysis of hisD2-nif cotransduction data by the Wu equation (Wu, 1966) suggested that the nif genes are divided into two clusters: a his-proximal cluster comprising nifBA(L)F and a his-distal group of nifEKDH.     

Site-directed mutagenesis of the binding site for ribosomal protein S8 within 16S ribosomal RNA from Escherichia coli.

Twelve specific alterations have been introduced into the binding site for ribosomal protein S8 in Escherichia coli 16S rRNA. Appropriate rDNA segments were first cloned into bacteriophage M13 vectors and subjected to bisulfite and oligonucleotide-directed mutagenesis in vitro. Subsequently, the mutagenized sequences were placed within the rrnB operon of plasmid pNO1301 and the mutant plasmids were used to transform E. coli recipients. The growth rates of cells containing the mutant plasmids were determined and compared with that of cells containing the wild-type plasmid. Only those mutations which occurred at highly conserved positions, or were expected to disrupt the secondary structure of the binding site, increased the doubling time appreciably. The most striking changes in growth rate resulted from mutations that altered a small internal loop within the S8 binding site. This structure is phylogenetically conserved in prokaryotic 16S rRNAs and may play a direct role in S8-16S rRNA recognition and interaction.     

Chloramphenicol resistance mutation in Escherichia coli which maps in the major ribosomal protein gene cluster.

Localized mutagenesis and selection for streptomycin resistance were utilized to isolate a chloramphenicol resistance mutation in Escherichia coli K-12 linked to the strA (rpsL) locus. Bacteriophage P1 transduction verified the map position of the new resistance mutation at 72 min, placing it within a dense cluster of ribosomal protein genes. The map position differs from that of known cmlA and cmlB mutations, which map at 18 and 21 min, respectively. Ribosomes prepared from chloramphenicol-resistant and -sensitive isogenic transductants were analyzed in vitro for activity in formation of N-formylmethionyl-puromycin, polyphenylalanine, and polylysine in the presence of inhibitory concentrations of chloramphenicol. Comparisons were also made of 14C-chloramphenicol binding to 70S ribosomes and of the two-dimensional polyacrylamide gel electrophoresis pattern of ribosomal proteins from each strain. There was no detectable difference between ribosomes from sensitive and resistant strains as measured by these assays. Enzymatic modification by chloramphenicol acetyltransferase is not responsible for the observed phenotype.     

Identification of Escherichia coli and Bacillus stearothermophilus ribosomal protein binding sites on Escherichia coli 5S RNA.

Summary E. coli [³²P]-labelled 5S RNA was complexed with E. coli and B. stearothermophilus 50S ribosomal proteins. Limited T₁ RNase digestion of each complex yielded three major fragments which were analysed for their sequences and rebinding of proteins. The primary binding sites for the E. coli binding proteins were determined to be sequences 18 to 57 for E-L5, 58 to 100 for E-L18 and 101 to 116 for E-L25. Rebinding experiments of purified E. coli proteins to the 5S RNA fragments led to the conclusion that E-L5 and E-L25 have secondary binding sites in the section 58 to 100, the primary binding site for E-L18. Since B. stearothermophilus proteins B-L5 and BL22 were found to interact with sequences 18 to 57 and 58 to 100 it was established that the thermophile proteins recognize and interact with RNA sequences similar to those of E. coli. Comparison of the E. coli 5S RNA sequence with those of other prokaryotic 5S RNAs reveals that the ribosomal proteins interact with the most conserved sections of the RNA.Paper number 12 on structure and function of 5S RNA.Preceding paper: Wrede, P. and Erdmann, V.A. Proc. Natl. Acad. Sci. USA 74, 2706–2709 (1977)     

Escherichia coli ribosomal protein L3 stimulates the helicase activity of the Bacillus stearothermophilus PcrA helicase.

Escherichia coli ribosomal protein L3 stimulates the in vitro helicase activity of Bacillus stearothermophilus PcrA helicase upon a variety of different substrates. L3 has no intrinsic helicase or ATPase activity nor is it able to stimulate the ATPase activity of PcrA. Gel mobility shift assays revealed that the affinity of PcrA for a variety of different DNA species (single-stranded, nicked and 3'-tailed) was enhanced in the presence of L3. We suggest that the stimulatory effect of L3 upon the helicase activity of PcrA is mediated via a protein-protein interaction which promotes cooperative binding of PcrA to its DNA substrate. This activity of L3 appears to be specific for PcrA helicase.     

Unbalanced ribosomal protein synthesis in a strain of Escherichia coli containing a cloned, truncated 16-S ribosomal RNA gene

An Escherichia coli K12 strain, carrying the promotor and proximal portion of the 16-S rRNA gene from rrnB cloned in the high-copy-number plasmid psF2124, has been examined for abnormalities in ribosome biogenesis. Both ribosomal RNA accumulation and ribosome content are depressed in this strain as compared to the control strain carrying the plasmid vector alone. The rate of total protein synthesis, however, appears to be normal. In contrast, the rate of ribosomal protein synthesis, relative to total protein synthesis, is elevated. The rates of synthesis of individual ribosomal proteins were determined and found to vary greatly, ranging from severe under-synthesis (displayed especially by proteins L7/L12) to massive over-synthesis (displayed particularly in the case of protein S7). Analysis of the rates of synthesis of other proteins coded for by the S12 operon revealed that protein S12 was moderately over-produced, but elongation factors EF-G and EF-Tu appear to be synthesized at the same rate as EF-Ts, all three being moderately under-synthesized relative to total soluble proteins.     

A missense mutation in the gene coding for ribosomal protein S17 (rpsQ) leading to ribosomal assembly defectivity in Escherichia coli.

Summary The conditionally lethal mutation, 286lmis, has been mapped inside the ribosomal protein gene cluster at 72 minutes on the Escherichia coli chromosome and was found to cotransduce at 97% with rpsE (S5). The 2861mis mutation leads to thermosensitivity and impaired assembly in vivo of 30S ribosomal particles at 42°C. The strain carrying the mutation has an altered S17 ribosomal protein; the mutational alteration involves a replacement of serine by phenylalanine in protein S17. Spontaneous reversion to temperature independence can restore the normal assembly in vivo of 30S ribosomal subunits at 42°C and the normal chromatographical sehaviour of the S17 ribosomal protein in vitro. We conclude therefore that the 2861mis mutation affects the structural gene for protein S17 (rpsQ).     

Interaction of the Escherichia coli DEAD box protein DbpA with 23 S ribosomal RNA.

The Escherichia coli DEAD box protein DbpA is unique among the DEAD box family in that its ATPase activity is specifically stimulated by bacterial 23 S ribosomal RNA. We have analysed the interaction between DbpA and a specific region within 23 S rRNA (namely nucleotides 2508-2580) which stimulates full ATPase activity. Using electrophoretic mobility shift assays we show that DbpA binds to this "specific" region with greater efficiency than to other regions of 23 S rRNA, and is not competed off by a non-specific RNA or a mutant RNA in which one of the stem-loops has been disrupted. These data suggest that the secondary structure within this region of 23 S rRNA is important for its recognition and binding by DbpA. We have also examined the ability of DbpA to unwind RNA and show that the purified protein does not behave as an RNA helicase in vitro with the substrates tested.