Marianne Grunberg-Manago

Enzymic synthesis of polynucleotides I. polynucleotide phosphorylase of Azotobacter vinelandii

The isolation, partial purification and some properties of polynucleotide phosphorylase of Azotobacter vinelandii are described. The enzyme catalyzes the synthesis of highly polymerized ribonucleic acid-like polynucleotides from 5′-nucleoside diphosphates with release of orthophosphate. The reaction requires magnesium ions and is reversible. Thus, the enzyme also catalyzes the phosphorolysis of polynucleotides to yield the corresponding 5′-nucleoside diphosphates. The preparation and isolation of a number of polynucleotides containing one or several kinds of mononucleotide units is described. The scope, mechanism and significance of the reaction are discussed.

Cleavage by RNase III in the transcripts of the metY-nusA-infB operon of Escherichia coli releases the tRNA and initiates the decay of the downstream mRNA☆

The metY gene coding for a minor form of the initiator tRNA is the first gene of a complex polycistronic operon also encoding the transcription termination factor NusA and the translation initiation factor IF2. The mixed tRNA-mRNA polycistronic transcript is cleaved by RNase III in a hairpin structure downstream from the tRNA. This cleavage separates the tRNA from the mRNA and initiates the rapid degradation of the 5 extremity of the downstream mRNA. Dissociation of the structural (tRNA) and informational (mRNA) RNAs from this operon is also achieved by independent transcription in vivo. The presence of two transcription terminators located downstream from metY produces a small tRNAMetf2 precursor transcript, whereas an internal promoter situated between metY and the first open reading frame directs the transcription of only the protein-coding part of the operon.

Target Site of Escherichia coli Ribosomal Protein S15 on its Messenger RNA Conformation and Interaction with the Protein

The regulatory site of ribosomal protein S15 has been located in the 5 non-coding region of the messenger, overlapping with the ribosome loading site. The conformation of an in vitro synthesized mRNA fragment, covering the 105 nucleotides upstream from the initiation codon and the four first codons of protein S15, has been monitored using chemical probes and RNase V1. Our results show that the RNA is organized into three domains. Domains I and II, located in the 5 part of the mRNA transcript, are folded into stable stem-loop structures. The 3-terminal domain (III), which contains the Shine-Dalgarno sequence and the AUG initiation codon, appears to adopt alternative conformations. One of them corresponds to a rather unstable stem-loop structure in which the Shine-Dalgarno sequence is paired. An alternative potential structure involves a pseudo-knot interaction between bases of this domain and bases in the loop of domain II. The conformation of several RNA variants has also been investigated. The deletion of the 5-proximal stem-loop structure (domain I), which has no effect on the regulation, does not perturb the conformation of the two other domains. The deletion of domain II, leading to a loss of regulatory control, prevents the formation of the potential helix involved in the pseudo-knot structure and results in a stabilization of the alternative stem-loop structure in domain III. The replacement of another base in domain III involved in pairing in the two alternative structures mentioned above should induce a destabilization of both structures and results in a loss of the translational control. However, the replacement of another base in domain III, which does not abolish the control, results in the loss of the conformational heterogeneity in this domain and yields a stable conformation corresponding to the pseudo-knot structure. Thus, it appears that any mutation that disrupts or alters the formation of the pseudo-knot impairs the regulatory mechanism. Footprinting experiments show that protein S15 is able to bind to the synthesized fragment and provide evidence that the protein triggers the formation of the pseudo-knot conformation. A mechanism can be postulated in which the regulatory protein stabilizes this particular structure, thus impeding ribosome initiation.

Escherichia coli threonyl-tRNA synthetase and tRNAThr modulate the binding of the ribosome to the translational initiation site of the ThrS mRNA

Escherichia coli threonyl-tRNA synthetase binds to the leader region of its own mRNA at two major sites: the first shares some analogy with the anticodon arm of several tRNA(Thr) isoacceptors and the second corresponds to a stable stem-loop structure upstream from the first one. The binding of the enzyme to its mRNA target site represses its translation by preventing the ribosome from binding to its attachment site. The enzyme is still able to bind to derepressed mRNA mutants resulting from single substitutions in the anticodon-like arm. This binding is restricted to the stem-loop structure of the second site. However, the interaction of the enzyme with this site fails to occlude ribosome binding. tRNA(Thr) is able to displace the wild-type mRNA from the enzyme at both sites and suppresses the inhibitory effect of the synthetase on the formation of the translational initiation complex. Our results show that tRNA(Thr) acts as an antirepressor on the synthesis of its cognate aminoacyl-tRNA synthetase. This repression/derepression double control allows precise adjustment of the rate of synthesis of threonyl-tRNA synthetase to the tRNA level in the cell.

Aminoacyl‐tRNA synthetase gene regulation in Bacillus subtilis: induction, repression and growth‐rate regulation

The thrS gene in Bacillus subtilis is specifically induced by starvation for threonine and is, in addition, autorepressed by the overproduction of its own gene product, the threonyl‐tRNA synthetase. Both methods of regulation employ an antitermination mechanism at a factor‐independent transcription terminator that occurs just upstream of the start codon. The effector of the induction mechanism is thought to be the uncharged tRNAThr, which has been proposed to base pair in two places with the leader mRNA to induce antitermination. Here we show that the autoregulation by synthetase overproduction is likely to utilize a mechanism similar to that characterized for induction by amino acid starvation, that is by altering the levels of tRNA charging in the cell. We also demonstrate that the base pairing interaction at the two proposed contact points between the tRNA and the leader are necessary but not always sufficient for either form of regulation. Finally, we present evidence that the thrS gene is expressed in direct proportion to the growth rate. This method of regulation is also at the level of antitermination but is independent of the interaction of the tRNA with the leader region.

A severely truncated form of translational initiation factor 2 supports growth of Escherichia coli

We have constructed strains carrying null mutations in the chromosomal copy of the gene for translational initiation factor (IF) 2 (infB). A functional copy of the infB gene is supplied in trans by a thermosensitive lysogenic lambda phage integrated at att lambda. These strains enabled us to test in vivo the importance of different structural elements of IF2 expressed from genetically engineered plasmid constructs. We found that, as expected, the gene for IF2 is essential. However, a protein consisting of the C-terminal 55,000 Mr fragment of the wild-type IF2 protein is sufficient to allow growth when supplied in excess. This result suggests that the catalytic properties are localized in the C-terminal half of the protein, which includes the G-domain, and that this fragment is sufficient to complement the IF2 deficiency in the infB deletion strain.

Identification of the gene encoding the 5S ribosomal RNA maturase in Bacillus subtilis: mature 5S rRNA is dispensable for ribosome function.

Over 25 years ago, Pace and coworkers described an activity called RNase M5 in Bacillus subtilis cell extracts responsible for 5S ribosomal RNA maturation (Sogin & Pace, Nature, 1974, 252:598-600). Here we show that RNase M5 is encoded by a gene of previously unknown function that is highly conserved among the low G + C gram-positive bacteria. We propose that the gene be named rnmV. The rnmV gene is nonessential. B. subtilis strains lacking RNase M5 do not make mature 5S rRNA, indicating that this process is not necessary for ribosome function. 5S rRNA precursors can, however, be found in both free and translating ribosomes. In contrast to RNase E, which cleaves the Escherichia coli 5S precursor in a single-stranded region, which is then trimmed to yield mature 5S RNA, RNase M5 cleaves the B. subtilis equivalent in a double-stranded region to yield mature 5S rRNA in one step. For the most part, eubacteria contain one or the other system for 5S rRNA production, with an imperfect division along gram-negative and gram-positive lines. A potential correlation between the presence of RNase E or RNase M5 and the single- or double-stranded nature of the predicted cleavage sites is explored.

Physical localisation and cloning of the structural gene for E. coli initiation factor IF3 from a group of genes concerned with translation

The structural genes for translational initiation factor IF3, threonyl-tRNA synthetase (TRS), the two subunits of phenylalanyl-tRNA synthetase (PRS), and a 12 000 mol. wt. protein of unidentified function are carried by the lambda p2 transducing phage. The localization of these genes on a restriction map of the Escherichia coli DNA insert was achieved by deletion mapping. In addition a set of plasmids carrying fragments of the original phage was constructed and helped to confirm these assignments. One plasmid, containing a 3.3 kb PstI fragment inserted into pBR322, does not code for any of the synthetase genes but causes strains carrying it to overproduce IF3.

Translational autocontrol of the Escherichia coli ribosomal protein S15

When rpsO, the gene encoding the ribosomal protein S15 in Escherichia coli, is carried by a multicopy plasmid, the mRNA synthesis rate of S15 increases with the gene dosage but the rate of synthesis of S15 does not rise. A translational fusion between S15 and beta-galactosidase was introduced on the chromosome in a delta lac strain and the expression of beta-galactosidase studied under different conditions. The presence of S15 in trans represses the beta-galactosidase level five- to sixfold, while the synthesis rate of the S15-beta-galactosidase mRNA decreases by only 30 to 50%. These data indicate that S15 is subject to autogenous translational control. Derepressed mutants were isolated and sequenced. All the point mutations map in the second codon of S15, suggesting a location for the operator site that is very near to the translation initiation codon. However, the creation of deletion mutations shows that the operator extends into the 5 non-coding part of the message, thus overlapping the ribosome loading site.

Yeast lysyl-tRNA synthetase: Complex formation and heat protection by substrates

Abstract Complex formation between lysyl-tRNA synthetase and its substrates was investigated. A ratio of 1:1:1 of tRNA:AMP-lysine:synthetase was obtained, based on an assumed molecular weight of 112 000 for the enzyme. The enzyme was found to be thermolabile with no charging activity remaining after 2 min at 40° in the presence of 1 mM Mg2+. In the absence of Mg2+ sensitivity was decreased. It is protected against heat inactivation by tRNA, poly U, poly C, ATP, ATP+lysine, and lysinol-AMP, while lysine alone has no protective effect. An enzyme-ATP complex was isolated which could transfer lysine to tRNA.