Control of ribosomal RNA synthesis in Escherichia coli

Summary The ribosomal RNA synthesis in a cell-free system containing the nucleoids and the cytoplasmic fraction prepared from Escherichia coli cells has been investigated. The addition of the 4S fraction from the cytoplasm to the isolated nucleoids induces RNA synthesis by a new chain initiation. In this system a preferential initiation of rRNA chains occurs. The experimental results suggest that the 4S fraction contains at least two activities, one for releasing RNA-polymerases from the nucleoids, and another for the frequent initiation of rRNA chains. No restriction of the rRNA synthesis has been observed in the nucleoids and the 4S fraction from the amino acidstarved rel ⁺ cells. The rRNA synthesized in the above system is detected at about 23S and 16S rRNA regions.     

The stoichiometry of the ribosomal proteins of Escherichia coli

Summary A ribosome preparation from E. coli made without stringent washing procedures has been shown to contain the same relative amounts of nearly all the ribosomal proteins as ribosomes in intact cells. Stoichiometric measurements on all the proteins of this preparation except for L8, L20, L31 and L34 have been made using an isotope dilution technique. When the scatter of the values obtained, the uncertainty in the molecular weights, and the losses occurring during extraction are taken into account, none of the proteins except L7/L12 is present at a level significantly different from one molecule per ribosome. There are multiple copies of L7/L12. These data suggest that the ribosomes of Escherichia coli are homogeneous in vivo.     

Homology between Drosophila melanogaster and Escherichia coli ribosomal proteins

Summary Antibodies raised against D. melanogaster ribosomal proteins were used to examine possible structural relationships between eukaryotic and prokaryotic ribosomal proteins. The antisera were raised against either groups of ribosomal proteins or purified individual ribosomal proteins from D. melanogaster. The specificity of each antiserum was confirmed and the identity of the homologous E. coli ribosomal protein was determined by immunochemical methods. Immuno-overlay assays indicated that the antiserum against the D. melanogaster small subunit protein S14 (anti-S14) was highly specific for protein S14. In addition, anti-S14 showed a cross-reaction with total E. coli ribosomal proteins in Ouchterlony double immunodiffusion assays and with only E. coli protein S6 in immuno-overlay assays. From these and other experiments with adsorption of anti-S14 with individual purified proteins, the E. coli protein homologous to the D. melanogaster protein S14 was established as protein S6.     

Genetic studies of the ribosomal proteins in Escherichia coli X

Summary Episomes of E. coli, which cover argG but not the str region, were transferred to Serratia marcescens. Ribosomal proteins from these hybrid strains were analyzed with phospho-cellulose or carboxymethyl-cellulose column chromatography. Two E. coli ribosomal proteins, L21 and S15, could be detected in the ribosome from the hybrid strains in addition to the ribosomal proteins of S. marcescens.     

Methylation of ribosomal proteins during ribosome assembly in Escherichia coli

Summary In Escherichia coli, a number of ribosomal proteins are methylated. The time of methylation of L7 and L11 during ribosome assembly was studied. It was observed that the methylation of L7 could occur in the free protein stage. Both the 32S and 40S ribonucleoprotein intermediates also contained methylated L7 although the extent of methylation in these particles was not as high as in the free L7, the 45S or the 50S particles. Free L11 could also be partially methylated but the bulk of methylation of this protein was found in the 45S and the 50S particles.It was previously reported that the methylation of L7 is inversely proportional to the growth temperature (Chang 1978), we now show that once L7 is methylated at 25°, the methyl group is stable when the culture is shifted to 37°C. However, a partial turnover of the methyl group of L7 is observed when the methylated ribosome is chased at 25°C. On the other hand, the methyl groups of L11 appear to be stable at either 25°C or 37°C. We also observe that the extent of methylation of both L7 and L11 stays nearly constant during the cell growth cycle from early log to stationary phase.     

The hinge region of Escherichia coli ribosomal protein L7/L12 is required for factor binding and GTP hydrolysis

A variant form of Escherichia coli ribosomal protein L7/L12 that lacked residues 42 to 52 (L7/L12 Δ42–52) in the hinge region was shown previously to be completely inactive in supporting polyphenylalanine synthesis although it bound to L7/L12 deficient core particles with the normal stoichiometry of four copies per particle (Oleinikov AV, Perroud B, Wang B, Traut RR (1993) J Biol Chem, 268, 917–922). The result suggested that the hinge confers flexibility that is required for activity because the resulting bent conformation allows the distal C-terminal domain to occupy a location on the body of the large ribosomal subunit proximal to the base of the L7/L12 stalk where elongation factors bind. Factor binding to the hinge-truncated variant was tested. As an alternative strategy to deleting residues from the hinge, seven amino acid residues within the putative hinge region were replaced by seven consecutive proline residues in an attempt to confer increased rigidity that might reduce or eliminate the bending of the molecule inferred to be functionally important. This variant, L7/L12: (Pro)₇, remained fully active in protein synthesis. Whereas the binding of both factors in ribosomes containing L7/L12:Δ42–52 was decreased by about 50%, there was no loss of factor binding in ribosomes containing L7/L12:(Pro)₇, as predicted from the retention of protein synthesis activity. The factor:ribosome complexes that contained L7/L12:Δ42–52 had the same low level of GTP hydrolysis as the core particles completely lacking L7/L12 and EF-G did not support translocation measured by the reaction of phe-tRNA bounds in hr Asite with puromycin. It is concluded that the hinge region is required for the functionally productive binding of elongation factors, and the defect in protein synthesis reported previously is due to this defect. The variant produced by the introduction of the putative rigid Pro₇ sequence retains sufficient flexibility for full activity.     

Cooperative control of translational fidelity by ribosomal proteins in Escherichia coli

Summary The effect on translational fidelity of a particular mutation in the gene coding for protein S5 (rpxE) has been investigated. This mutation has the opposite effect of a restrictive strA mutation; in vivo, it relieves the restriction imposed by strA on the suppression of T4 nonsense mutants and results in hypersensitivity to streptomycin; in vitro, the presence of the altered S5 protein in 30S ribosomes results in increased intrinsic misreading. It is concluded that this mutation, ramC₃₁₉, acts as a ribosomal ambiguity mutation similar to certain mutations of protein S4 (ramA).     

Cooperative control of translational fidelity by ribosomal proteins in Escherichia coli

Summary Two spontaneous mutants of Escherichia coli strain KMBL-146 selected for resistance to the aminoglycoside antibiotic neamine show severe restriction of amber suppressors in vivo. Purified ribosomes from the mutant strains exhibit low neamine-induced misreading in vitro and a decreased affinity for the related antibiotic streptomycin.Biochemical analysis shows that the mutants each have two modified 30S ribosomal proteins, S12 and S5. In agreement with these results, genetic analysis shows that two mutations are present, neither of which confers resistance to neamine by itself; the mutation located in gene rpxL (the structural gene for protein S12) confers streptomycin dependence but this dependence is suppressed in the presence of the second mutation, located in gene rpxE (the structural gene for protein S5).     

Isolation and characterization of temperature-sensitive mutants of Escherichia coli with altered ribosomal proteins

Summary The ribosomal proteins of temperature-sensitive mutants of Escherichia coli isolated independently after mutagenesis with nitrosoguanidine were analyzed by two-dimensional gel electrophoresis. Out of 400 mutants analyzed, 60 mutants (15%) showed alterations in a total of 22 different ribosomal proteins. The proteins altered in these mutants are S2, S4, S6, S7, S8, S10, S15, S16, S18, L1, L3, L6, L10, L11, L14, L15, L17, L18, L19, L22, L23 and L24. A large number of them (25 mutants) have mutations in protein S4 of the small subunit, while four mutants showed alterations in protein L6 of the large subunit. The importance of these mutants for structural and functional analyses of ribosomes is discussed.     

Revised location of the Escherichia coli gene coding for ribosomal proteins S2

Summary P1 transduction studies of a mutation of Escherichia coli affecting the rpsB gene previously suggested the map order popC-rpsB-dapD. Biochemical analysis of transductants has now revealed that the rpsB gene lies in fact to the right hand (tsf) side of the dapD marker.     

Immunological studies of Escherichia coli mutants lacking one or two ribosomal proteins

Summary A battery of immunological tests were used to investigate mutants which had been determined as lacking one or two ribosomal proteins on the basis of two-dimensional polyacrylamide gels. Proteins which were confirmed as missing from the ribosome in one or more mutants were large subunit proteins L1, L15, L19, L24, L27, L28, L30 and L33 and small subunit proteins S1, S9, S17 and S20. Cross-reacting material (CRM) was also absent from the post-ribosomal supernatant except in the case of protein S1. Since mutants lacking protein L11 have been previously described, any one of 13 of the 52 ribosomal proteins can be missing. None of these 13 proteins, except S1, can therefore have an indispensable role in ribosome function or assembly. In several mutants in which a protein was not missing but altered, it was present as several moieties of differing charge and size.     

Stoichiometric measurements of 30S and 50S ribosomal proteins from Escherichia coli

Summary Stoichiometric investigations were made for all 21 proteins of the 30S subunit and for 31 proteins of the 50S subunit. While the results for the 30S proteins strongly suggest the existence of two different stoichiometric classes, the so-called fractional (F) and unit (U) proteins with average molar amounts of 0.1–0.5 and 0.8–1.2 per mole 30S respectively, at least one further group is present in isolated 50S particles. Therefore 50S proteins may be subdivided in the following way: a) 9–10 proteins with average molar amounts of 0.2–0.6 (F-proteins), b) 15–16 proteins with 0.8–1.2 copies per 50S particle (U-proteins), c) at least one protein with an average value of 1.8–2.2 copies, the repeated (R) proteins, d) a group of 7 proteins with 1.4–1.7 copies per particle; they can be named fractional repeated (FR) proteins. These findings indicate that 50S ribosomal particles show a more differentiated degree of structural heterogeneity at least in vitro than has been shown for 30S subunits.Paper No. 35 on Ribosomal Proteins. Preceding paper is by E. Deusser and H. G. Wittmann, Nature 238, 269 (1972).     

Expression of the L11-L1 operon in mutants of Escherichia coli lacking the ribosomal proteins L1 or L11

Summary Using a variety of immunological techniques, the supernatant levels of ribosomal proteins were measured in mutants lacking the ribosomal proteins L1 or L11, and in wild-type strains. There was a 2.5–5-fold elevation of protein L11 level in the supernatant of strains lacking protein L1, compared to wild-type. In contrast, there was no elevation, but rather a diminution, in the corresponding L1 level in strains lacking protein L11, compared to wild-type. These results are consistent with a model for the control of expression of the L11-L1 operon in which protein L1 is an inhibitor of expression of that operon, but protein L11 is not. The supernatant concentrations of other proteins were indistinguishable in all strains.     

Single-stranded conjugation in Escherichia coli K-12

Summary The mutation BT43 in the gene dnaB leads to the inhibition of vegetative and conjugational DNA synthesis at 42°. The consequences in case of conjugation are very unusual. The fragment of donor DNA tramsmitted to the recipient cell remains single-stranded and is integrated as such into the recipient chromosome similar to the main events during transformation. We call this process single-stranded (SS) conjugation.The evidence for this statement comes from the measurement of the time of expression of the gene tsx, containing the genetic information for the receptor of phage T6. The gene tsx is introduced into a dnaBT43 recipient cell alternatively by two different donors Hfr H and Hfr C, which are characterized by opposite directions of transfer. Therefore both donors introduce into the recipient cell alternatively the informational or noninformational DNA strand. If conjugation is performed at a nonpermissive temperature, the transferred DNA piece remains single-stranded and is integrated as such into the recipient chromosome. If it is the informational strand (case of Hfr H), it is transcribed very fast and yields the protein in question in about 20 min. If the noninformational strand is integrated (Hfr C) about 40 min additional time is required to effect cell division.SS-conjugation is very sensitive to the action of exonucleases Exo I and Exo V and is much enhanced in the absence of both nucleases in the recipient.The exogenous DNA pieces are integrated as short insertions, this leads to the disjoining of linked markers and to a very short scale of the genetic map. Because the donor DNA undergoes recombination in the single-stranded state heteroduplex regions originate which are subsequently corrected by the enzymes of the recipient cell. The situation leads to a very special but predictable heterogeneity of the progeny of transconjugants.The fact of the existence of this special process, SS-conjugation, drastically different from common conjugation in many respects, suggests that common conjugation leads to the integration of double-stranded DNA pieces into the recipient chromosome.     

Localization of the structural gene for ribosomal protein L19 (rplS) in Escherichia coli

Summary A temperature-sensitive mutant derived from an E. coli K12 strain, PA3092, was found to have an alteration in the ribosomal protein L19 (Isono et al., 1977). This mutant is a double mutant with a temperature-sensitivity mutation and a mutation leading to the structural alteration of L19 protein. Crosses with various Hfr strains and transductions with P1kc have revealed that the latter mutation maps at 56.4 min, between pheA and alaS. From the fact that two other mutations causing different types of alterations in L19 protein also map at this locus, the gene affected by these mutations was concluded to be the structural gene for the ribosomal protein L19 (rplS).