A key role for the mRNA leader structure in translational control of ribosomal protein S1 synthesis in gamma-proteobacteria

The translation initiation region (TIR) of the Escherichia coli rpsA mRNA coding for ribosomal protein S1 is characterized by a remarkable efficiency in driving protein synthesis despite the absence of the canonical Shine–Dalgarno element, and by a strong and specific autogenous repression in the presence of free S1 in trans. The efficient and autoregulated E.coli rpsA TIR comprises not less than 90 nt upstream of the translation start and can be unambiguously folded into three irregular hairpins (HI, HII and HIII) separated by A/U-rich single-stranded regions (ss1 and ss2). Phylogenetic comparison revealed that this specific fold is highly conserved in the γ-subdivision of proteobacteria (but not in other subdivisions), except for the Pseudomonas group. To test phylogenetic predictions experimentally, we have generated rpsA′–′lacZ translational fusions by inserting the rpsA TIRs from various γ-proteobacteria in-frame with the E.coli chromosomal lacZ gene. Measurements of their translation efficiency and negative regulation by excess protein S1 in trans have shown that only those rpsA TIRs which share the structural features with that of E.coli can govern efficient and regulated translation. We conclude that the E.coli-like mechanism for controlling the efficiency of protein S1 synthesis evolved after divergence of Pseudomona     

An amber mutation in the gene rpsA for ribosomal protein S1 in Escherichia coli

Summary An amber mutation has been induced in the gene rpsA (which codes fo ribosomal protein S1) of Escherichia coli K-12 strain in the presence of an amber suppressor (supD) and mutations sueA, sueB and sueC that additively enhance the efficiency of suppression. That the amber mutation has occurred in the gene rpsA was confirmed by complementation with a plasmid which carried the wild-type allele of rpsA. The mutation is lethal in the absence of an amber suppressor, indicating that ribosomal protein S1 is indispensable to E. coli.     

Crystal structure of Escherichia coli PNPase: central channel residues are involved in processive RNA degradation

Bacterial polynucleotide phosphorylase (PNPase) plays a major role in mRNA turnover by the degradation of RNA from the 3′- to 5′-ends. Here, we determined the crystal structures of the wild-type and a C-terminal KH/S1 domain-truncated mutant (ΔKH/S1) of Escherichia coli PNPase at resolutions of 2.6 Å and 2.8 Å, respectively. The six RNase PH domains of the trimeric PNPase assemble into a ring-like structure containing a central channel. The truncated mutant ΔKH/S1 bound and cleaved RNA less efficiently with an eightfold reduced binding affinity. Thermal melting and acid-induced trimer dissociation studies, analyzed by circular dichroism and dynamic light scattering, further showed that ΔKH/S1 formed a less stable trimer than the full-length PNPase. The crystal structure of ΔKH/S1 is more expanded, containing a slightly wider central channel than that of the wild-type PNPase, suggesting that the KH/S1 domain helps PNPase to assemble into a more compact trimer, and it regulates the channel size allosterically. Moreover, site-directed mutagenesis of several arginine residues in the channel neck regions produced defective PNPases that either bound and cleaved RNA less efficiently or generated longer cleaved oligonucleotide products, indicating that these arginines were involved in RNA binding and processive degradation. Taking these results together, we conclude that the constricted central channel and the basic-charged residues in the channel necks of PNPase play crucial roles in trapping RNA for processive exonucleolytic degradation.     

RNR1, a 3'-5' exoribonuclease belonging to the RNR superfamily, catalyzes 3' maturation of chloroplast ribosomal RNAs in Arabidopsis thaliana

Arabidopsis thaliana chloroplasts contain at least two 3′ to 5′ exoribonucleases, polynucleotide phosphorylase (PNPase) and an RNase R homolog (RNR1). PNPase has been implicated in both mRNA and 23S rRNA 3′ processing. However, the observed maturation defects do not affect chloroplast translation, suggesting that the overall role of PNPase in maturation of chloroplast rRNA is not essential. Here, we show that this role can be largely ascribed to RNR1, for which homozygous mutants germinate only on sucrose-containing media, and have white cotyledons and pale green rosette leaves. Accumulation of chloroplast-encoded mRNAs and tRNAs is unaffected in such mutants, suggesting that RNR1 activity is either unnecessary or redundant for their processing and turnover. However, accumulation of several chloroplast rRNA species is severely affected. High-resolution RNA gel blot analysis, and mapping of 5′ and 3′ ends, revealed that RNR1 is involved in the maturation of 23S, 16S and 5S rRNAs. The 3′ extensions of the accumulating 5S rRNA precursors can be efficiently removed in vitro by purified RNR1, consistent with this view. Our data suggest that decreased accumulation of mature chloroplast ribosomal RNAs leads to a reduction in the number of translating ribosomes, ultimately compromising chloroplast protein abundance and thus plant growth and development.     

The POL1 gene from the fission yeast, Schizosaccharomyces pombe, shows conserved amino acid blocks specific for eukaryotic DNA polymerases alpha

Summary The POL1 gene of the fission yeast, Schizosaccharomyces pombe, was isolated using a POL1 gene probe from the budding yeast Saccharomyces cerevisiae, cloned and sequenced. This gene is unique and located on chromosome II. It includes a single 91 by intron and is transcribed into a mRNA of about 4500 nucleotides. The predicted protein coded for by the S. pombe POL1 gene is 1405 amino acid long and its calculated molecular weight is about 160000 daltons. This peptide contains seven amino acid blocks conserved among several DNA polymerases from different organisms and shares overall 37% and 34% identity with DNA polymerases alpha from S. cerevisiae and human cells, respectively. These results indicate that this gene codes for the S. pombe catalytic subunit of DNA polymerase alpha. The comparisons with human DNA polymerase alpha and with the budding yeast DNA polymerases alpha, delta and epsilon reveal conserved blocks of amino acids which are structurally and/or functionally specific only for eukaryotic alpha-type DNA polymerases.     

Conserved domains in polynucleotide phosphorylase among eubacteria

Polynucleotide phosphorylase (PNPase) is a polynucleotide nucleotidyl transferase (E. C. 2.7.7.8) that is involved in mRNA degradation in prokaryotes. PNPase structure analysis has been performed in Streptomyces antibioticus; this revealed the presence of five domains: two ribonuclease PH (RPH)-like (pnp1 and pnp2), one alpha helical, one KH, and one S1 domains. The trimeric nature of this enzyme was also confirmed. In this work, we have investigated conserved domains or subdomains in bacterial PNPases (55), for this structure-based sequence homology analysis between predicted amino acid sequences from bacterial PNPases and that of S. antibioticus was performed. Our findings indicate that while pnp2 (% similarity average S = 84/% identity average I = 22), KH (S = 74.3%/I = 5.3%), S1 (S = 71.3%/I = 1.2%); and pnp1 (S = 52.8%/I = 0.3%) domain; structure and sequence are well conserved among different bacteria, alpha helical domain (S = 39.5%/I = 0) although conservation of the structure is somewhat maintained, the sequence is not conserved at all. Implications of such findings in PNPase activity will be discussed.     

Kinetics of polynucleotide phosphorylase: comparison of enzymes from Streptomyces and Escherichia coli and effects of nucleoside diphosphates

We examined the activity of polynucleotide phosphorylase (PNPase) from Streptomyces coelicolor, Streptomyces antibioticus, and Escherichia coli in phosphorolysis using substrates derived from the rpsO-pnp operon of S. coelicolor. The Streptomyces and E. coli enzymes were both able to digest a substrate with a 3′ single-stranded tail although E. coli PNPase was more effective in digesting this substrate than were the Streptomyces enzymes. The k_cat for the E. coli enzyme was ca. twofold higher than that observed with the S. coelicolor enzyme. S. coelicolor PNPase was more effective than its E. coli counterpart in digesting a substrate possessing a 3′ stem-loop structure, and the K_m for the E. coli enzyme was ca. twice that of the S. coelicolor enzyme. Electrophoretic mobility shift assays revealed an increased affinity of S. coelicolor PNPase for the substrate possessing a 3′ stem-loop structure compared with the E. coli enzyme. We observed an effect of nucleoside diphosphates on the activity of the S. coelicolor PNPase but not the E. coli enzyme. In the presence of a mixture of 20 μM ADP, CDP, GDP, and UDP, the K_m for the phosphorolysis of the substrate with the 3′ stem-loop was some fivefold lower than the value observed in the absence of nucleoside diphosphates. No effect of nucleoside diphosphates on the phosphorolytic activity of E. coli PNPase was observed. To our knowledge, this is the first demonstration of an effect of nucleoside diphosphates, the normal substrates for polymerization by PNPase, on the phosphorolytic activity of that enzyme.     

1,2‐Diacylglycerol choline phosphotransferase catalyzes the final step in the unique Treponema denticola phosphatidylcholine biosynthesis pathway

Treponema denticola synthesizes phosphatidylcholine through a licCA‐dependent CDP‐choline pathway identified only in the genus Treponema. However, the mechanism of conversion of CDP‐choline to phosphatidylcholine remained unclear. We report here characterization of TDE0021 (herein designated cpt) encoding a 1,2‐diacylglycerol choline phosphotransferase homologous to choline phosphotransferases that catalyze the final step of the highly conserved Kennedy pathway for phosphatidylcholine synthesis in eukaryotes. T. denticola Cpt catalyzed in vitro phosphatidylcholine formation from CDP‐choline and diacylglycerol, and full activity required divalent manganese. Allelic replacement mutagenesis of cpt in T. denticola resulted in abrogation of phosphatidylcholine synthesis. T. denticola Cpt complemented a Saccharomyces cerevisiae CPT1 mutant, and expression of the entire T. denticola LicCA‐Cpt pathway in E. coli resulted in phosphatidylcholine biosynthesis. Our findings show that T. denticola possesses a unique phosphatidylcholine synthesis pathway combining conserved prokaryotic choline kinase and CTP:phosphocholine cytidylyltransferase activities with a 1,2‐diacylglycerol choline phosphotransferase that is common in eukaryotes. Other than in a subset of mammalian host‐associated Treponema that includes T. pallidum, this pathway is found in neither bacteria nor Archaea. Molecular dating analysis of the Cpt gene family suggests that a horizontal gene transfer event introduced this gene into an ancestral Treponema well after its divergence from other spirochetes.     

Mutational analysis of polynucleotide phosphorylase from Escherichia coli

Polynucleotide phosphorylase (PNPase), a homotrimeric exoribonuclease present in bacteria, is involved in mRNA degradation. In Escherichia coli, expression of this enzyme is autocontrolled at the translational level. We introduced about 30 mutations in the pnp gene by site-directed mutagenesis, most of them in phylogenetically conserved residues, and determined their effects on the three catalytic activities of PNPase, phosphorolysis, polymerisation and phosphate exchange, as well as on the efficiency of translational repression. The data are presented and discussed in the light of the crystallographic structure of PNPase from Streptomyces antibioticus. The results show that both PNPase activity and the presence of the KH and S1 RNA-binding domains are required for autocontrol. Deletions of these RNA-binding domains do not abolish any of the three catalytic activities, indicating that they are contained in a domain independent of the catalytic centre. Moreover, the catalytic centre was located around the tungsten-binding site identified by crystallography. Some mutations affect the three catalytic activities differently, an observation consistent with the presence of different subsites.