Leendert Bosch

RNA pseudoknots: Translational frameshifting and readthrough on viral RNAs

Ribosomal frameshifting on retroviral RNAs has been proposed to be mediated by slippage of two adjacent tRNAs into the — 1 direction at a specific heptanucleotide sequence. Here we report a computer-aided analysis of the structure around the established or putative frameshift sites in a number of retroviral, coronaviral, toroviral, and luteoviral RNAs and two dsRNA yeast viruses. In almost all cases a stable hairpin was predicted four to nine nucleotides downstream of the shifty heptanucleotide. More than half of the resulting hairpin loops give rise to potential pseudoknotting with sequences downstream of this hairpin. Especially in the case of the shifty heptanucleotides U UUA AAC and G GGA AAC, stable downstream pseudoknots are present. Indications were also found for the presence of pseudoknots downstream of amber stop condons at readthrough sites in some retroviral RNAs.

The role of FIS in trans activation of stable RNA operons of E. coli.

The thrU(tufB) operon of Escherichia coli is endowed with a cis‐acting region upstream of the promoter, designated UAS for Upstream Activator Sequence. A protein fraction has been isolated that binds specifically to DNA fragments of the UAS, thus forming three protein‐DNA complexes corresponding to three binding sites on the UAS. It stimulates in vitro transcription of the operon by facilitating the binding of the RNA polymerase to the promoter. All three protein‐DNA complexes contain one and the same protein. Dissociation constants for the three complexes have been determined, the lowest being in the sub‐nanomolar range. The protein also binds to the UAS of the tyrT operon and to the UAS upstream of the P1 promoter of the rrnB operon, suggesting that transcription of the three operons, if not of more stable RNA operons, is activated by a common trans activator. We demonstrate that the E.coli protein FIS (Factor for Inversion Stimulation) also binds to the UAS of the thrU(tufB) operon forming three protein‐DNA complexes. A burst of UAS‐ and FIS‐dependent promoter activity is observed after reinitiation of growth of stationary cultures in fresh medium.

Three-dimensional models of the tRNA-like 3' termini of some plant viral RNAs.

Various plant viral RNAs possess a 3′ terminus with tRNA‐like properties. These viral RNAs are charged with an amino acid upon incubation with the cognate aminoacyl‐tRNA synthetase and ATP. We have studied the structure of end‐labelled 3′‐terminal fragments of turnip yellow mosaic virus RNA and brome mosaic virus RNA 2 with chemical modifications of the adenosine and cytidine residues and with enzymatic digestions using RNase T1, nuclease S1 and the double‐strand‐specific ribonuclease from cobra venom. The data indicate that the 3′ termini of these plant viral RNAs lack a cloverleaf structure as found in classical tRNA. The three‐dimensional folding, however, reveals a striking resemblance with classical tRNA. The models proposed are supported by phylogenetic data. Apparently distinct three‐dimensional solutions have evolved to meet the requirements for faithful recognition by tRNA‐specific enzymes. The way in which the aminoacyl acceptor arms of these tRNA‐like structures are constructed reveal novel features in RNA folding which may have a bearing on the secondary and tertiary structures of RNA in general. The dynamic behaviour of brome mosaic virus RNA 2 in solution presumably is illustrative of conformational transitions, which RNAs generally undergo on changing the ionic conditions.

The Elongation Factor EF-Tu and Its Two Encoding Genes

Publisher Summary The chapter discusses all of the current knowledge concerning EF-Tu. It discusses some of the recent insights that emerge from an integration of genetic, biochemical, and biophysical studies of this interesting protein. The considerable amount of structural information regarding EF-Tu and its encoding genes and the construction of plasmids harboring these genes has opened new avenues toward an understanding of EF-Tu function at the molecular level. Structural alterations in the polypeptide chain have been brought about by mutagenesis; the functional consequences will be reviewed in this chapter. The availability of E.coli mutants, specifically altered in tufA or tufB or in both, also enabled the study of the regulation of the expression of these genes. These studies have led to the conclusion that the expression of tufA and tuft3 is coordinately regulated, but that two distinct mechanisms control the expression of the two genes. They have also indicated that EF-Tu itself is involved in the regulation of the expression of tufB, but not in that of tufA. The evidence supporting the hypothesis that EF-Tu acts as an autogenous repressor inhibiting tufB expression is discussed in this chapter.

In vitro translation of alfalfa mosaic virus RNA

RNA derived from the plant virus alfalfa mosaic virus (top component α) has been used as messenger RNA in cell-free systems of Escherichia coli . The radio-active polypeptides released from the ribosomes during incubation in the presence of [ 14 C]lysine, [ 14 C]arginine and non-radioactive amino acids, have been analysed. About 50% of the radioactivity is incorporated into polypeptides which coprecipitate with carrier viral coat protein in the presence of 0.05 m -magnesium sulphate. The precipitate, but not the soluble protein, shows a striking similarity to viral coat protein when studied by fingerprint analysis of its tryptic digest. Polypeptides synthesized under the direction of RNA from turnip yellow mosaic virus coprecipitate to a much lesser extent with alfalfa mosaic virus carrier protein in the presence of 0.05 m -magnesium sulphate and their tryptic peptides do not correspond to those of the latter viral coat protein.

The three-dimensional folding of the tRNA-like structure of tobacco mosaic virus RNA. A new building principle applied twice.

The structure of the tRNA‐like 3′ terminus of tobacco mosaic virus (TMV) RNA has been studied. A 3′ ‐terminal fragment possessing the tRNA‐like properties was probed with chemical modification and enzymatic digestions. A model of the secondary structure is proposed for the last 105 nucleotides. The corresponding region of other tobamoviral RNAs can be folded in an identical secondary structure. A three‐dimensional model for the tRNA‐like structure is given which is compared with those proposed earlier for the tRNA‐like 3′ termini of turnip yellow mosaic virus (TYMV) RNA and brome mosaic virus (BMV) RNA. A new building principle which we discovered previously by studying the latter RNAs appears to be applied twice in the tRNA‐like structure of TMV RNA. The determination of the minimal length requirement for recognition of CTP, ATP:tRNA nucleotidyl‐transferase reveals a size of ˜100 nucleotides in agreement with the models proposed.

Mutants of the elongation factor EF-Tu, a new class of nonsense suppressors.

Read‐through of nonsense codons has been studied in wild‐type Escherichia coli cells and in cells harbouring mutant species of the elongation factor EF‐Tu. The two phenomena differ essentially. Readthrough of UGA in wild‐type cells is reduced by inactivation of tufB but is restored to the original level by introducing into the cell plasmid‐borne EF‐Tu. This shows that the natural UGA leakiness is dependent on the intracellular concentration of EF‐Tu. Strains of E. coli harbouring mutant species of the elongation factor EF‐Tu suppress the nonsense codons UAG, UAA and UGA. Suppression shows a codon context dependence. It requires the combined action of two different EF‐Tu species: EF‐TuAR(Ala 375‐‐‐‐Thr) and EF‐TuBo(Gly 222‐‐‐‐Asp). Cells harbouring EF‐TuAR(Ala 375‐‐‐‐Thr) and wild‐type EF‐TuB, or wild‐type EF‐TuA and EF‐TuBo(Gly 222‐‐‐‐Asp) do not display suppressor activity. These data demonstrate that mutated tuf genes form an additional class of nonsense suppressors. The requirement for two different mutant EF‐Tu species raises the question whether translation of sense codons also occurs by the combined action of two EF‐Tu molecules on the ribosome.

Properties of the tobacco mosaic virus intermediate length RNA-2 and its translation

The existence of subgenomic RNAs is well established in the case of plant viruses such as tobacco mosaic virus (TMV). However, except for the subgenomic coat protein mRNA, it is not known whether the other subgenomic RNAs have a function in the life cycle of the virus. In search of more information about one of the major subgenomic RNAs-intermediate length RNA-2 or I2 RNA-of TMV, in vitro and in vivo translational studies were performed. The I2 RNA, which codes in vitro for the synthesis of a 30K (K = kilodalton) protein, appears to be uncapped as judged by the need of different in vitro translation conditions for the synthesis of this protein, compared to the conditions required for the synthesis of the 126K and 183K proteins coded by the capped genomic RNA. In vivo a protein migrating in the same position as the 30K protein synthesized in vitro can be detected in infected tobacco leaves. Since this protein occurs transiently early upon infection, whether it is virus-coded or virus-induced, it could have an early function during infection.

Three tuf-like genes in the kirromycin producer Streptomyces ramocissimus.

We have identified, cloned and sequenced three tuf-like genes from Streptomyces ramocissimus (Sr.), the producer of the antibiotic kirromycin which inhibits protein synthesis by binding the polypeptide chain elongation factor EF-Tu. The tuf-1 gene encodes a protein with 71% amino acid residues identical to the well characterized elongation factor Tu of Escherichia coli (Ec.EF-Tu). The genetic location of tuf-1 downstream of a fus homologue and the in vitro activity of Sr.EF-Tu1 show that tuf-1 encodes a genuine EF-Tu. The putative Sr.EF-Tu2 and Sr.EF-Tu3 proteins are 69% and 63% identical to Ec.EF-Tu. Homologues of tuf-1 and tuf-3 were detected in all five Streptomyces strains investigated, but tuf-2 was found in S. ramocissimus only. The three tuf genes were expressed in E. coli and used to produce polyclonal antibodies. Western blot analysis showed that Sr.EF-Tu1 was present at all times under kirromycin production conditions in submerged and surface-grown cultures of S. ramocissimus and in germinating spores. The expression of tuf-2 and tuf-3 was, however, below the detection level. Surprisingly, Sr.EF-Tu1 was kirromycin sensitive, which excludes the possibility that EF-Tu is involved in the kirromycin resistance of S. ramocissimus.

Composition of a methylated, acidic polysaccharide associated with coccoliths of Emiliania huxleyi (Lohmann) Kamptner

The water-soluble, acidic polysaccharide isolated from the coccoliths of the alga Emiliania huxleyi (Lohmann) Kamptner contains residues of the following sugars: l-galactose, d-glucose, d-mannose, l-mannose, l-rhamnose, l-arabinose, d-ribose, d-xylose, 6-O-methyl-d-mannose, 6-O-methyl-l-mannose, 2,3-di-O-methyl-l-rhamnose, 3-O-methyl-d-xylose, and d-galacturonic acid. l-Mannose, 6-O-methyl-d-mannose, 6-O-methyl-l-mannose, and 2,3-di-O-methyl-l-rhamnose are novel constituents of a polysaccharide. In addition, the presence of sulphate groups was found. Galacturonic acid and sulphate in the polysaccharide bind Ca2+ ions apparently in a ratio of one mol of Ca2+ per mol of acidic residue. This feature is relevant for the proposed matrix function of the polysaccharide in the formation of the calcified cell-wall plates (coccoliths) of the alga.