Insights into genome recoding from the mechanism of a classic +1-frameshifting tRNA

While genome recoding using quadruplet codons to incorporate non-proteinogenic amino acids is attractive for biotechnology and bioengineering purposes, the mechanism through which such codons are translated is poorly understood. Here we investigate translation of quadruplet codons by a +1-frameshifting tRNA, SufB2, that contains an extra nucleotide in its anticodon loop. Natural post-transcriptional modification of SufB2 in cells prevents it from frameshifting using a quadruplet-pairing mechanism such that it preferentially employs a triplet-slippage mechanism. We show that SufB2 uses triplet anticodon-codon pairing in the 0-frame to initially decode the quadruplet codon, but subsequently shifts to the +1-frame during tRNA-mRNA translocation. SufB2 frameshifting involves perturbation of an essential ribosome conformational change that facilitates tRNA-mRNA movements at a late stage of the translocation reaction. Our results provide a molecular mechanism for SufB2-induced +1 frameshifting and suggest that engineering of a specific ribosome conformational change can improve the efficiency of genome recoding.

Insights into Genome Recoding from the Mechanism of a Classic +1-Frameshifting tRNA

ABSTRACTWhile genome recoding using quadruplet codons to incorporate non-proteinogenic amino acids is attractive for biotechnology and bioengineering purposes, the mechanism through which such codons are translated is poorly understood. Here we investigate translation of quadruplet codons by a +1-frameshifting tRNA, SufB2, that contains an extra nucleotide in its anticodon loop. Natural post-transcriptional modification of SufB2 in cells prevents it from frameshifting using a quadruplet-pairing mechanism such that it preferentially employs a triplet-slippage mechanism. We show that SufB2 uses triplet anticodon-codon pairing in the 0-frame to initially decode the quadruplet codon, but subsequently shifts to the +1-frame during tRNA-mRNA translocation. SufB2 frameshifting involves perturbation of an essential ribosome conformational change that facilitates tRNA-mRNA movements at a late stage of the translocation reaction. Our results provide a molecular mechanism for SufB2-induced +1 frameshifting and suggest that engineering of a specific ribosome conformational change can improve the efficiency of genome recoding.

Loss of a Universal tRNA Feature

ABSTRACT tRNAHis has thus far always been found with one of the most distinctive of tRNA features, an extra 5′ nucleotide that is usually a guanylate. tRNAHis genes in a disjoint alphaproteobacterial group comprising the Rhizobiales, Rhodobacterales, Caulobacterales, Parvularculales, and Pelagibacter generally fail to encode this extra guanylate, unlike those of other alphaproteobacteria and bacteria in general. Rather than adding an extra 5′ guanylate posttranscriptionally as eukaryotes do, evidence is presented here that two of these species, Sinorhizobium meliloti and Caulobacter crescentus, simply lack any extra nucleotide on tRNAHis. This loss correlates with changes at the 3′ end sequence of tRNAHis and at many sites in histidyl-tRNA synthetase that might be expected to affect tRNAHis recognition, in the flipping loop, the insertion domain, the anticodon-binding domain, and the motif 2 loop. The altered tRNA charging system may have affected other tRNA charging systems in these bacteria; for example, a site in tRNAGlu sequences was found to covary with tRNAHis among alphaproteobacteria.

A New Biotype of Phaeosphaeria sp. of Uncertain Affinity Causing Stagonospora Leaf Blotch Disease in Cereals in Poland

A new Phaeosphaeria sp. biotype was isolated from winter ryes in Poland during 1995. Two isolates, Sn23-1 and Sn48-1, were obtained from diseased leaves of cvs. Motto and Dańkowskie, respectively. The rye Phaeosphaeria sp. represented by isolate Sn48-1 has similar pycnidiospore morphology and induces disease symptoms in cereals similar to Phaeosphaeria nodorum, the causal agent of Stagonospora nodorum blotch disease (4). The pathogen (Sn48-1) produces hyaline, cylindrical pycnidiospores that are mostly three-septate and measure 12.8 to 23.7 × 2.1 to 3.2 μm (average size = 16 × 2.6 μm) on water agar. A molecular comparison of several genes in isolates Sn23-1 and Sn48-1 revealed that the rye Phaeosphaeria sp. was different from P. nodorum. In the conserved alpha-box sequence (1,93 bp) of the MAT1-1 gene, a four nucleotide difference occurred between the wheat-biotype P. nodorum and isolates Sn23-1 and Sn48-1 (GenBank Accession Nos. AY072933 and AF322008). In addition, the length of the internal transcribed spacer (ITS) region of the nuclear rDNA was the same for the wheat-biotype P. nodorum and the two rye Phaeosphaeria sp. isolates. However, a six nucleotide discrepancy was found in the ITS region (GenBank Accession Nos. U77362 and AF321323). The beta-glucosidase (bgl1) and beta-tubulin (tubA) genes differ in length between the wheat-biotype P. nodorum and two rye Phaeosphaeria sp. isolates (2,3). The main difference was due to the intron sizes of these two genes. One extra nucleotide was found in the intron2 of the bgl1 gene (GenBank Accession Nos. AY683619 and AY683620) and the intron1 of the tubA gene (GenBank Accession Nos. AY786337 and AY786331), respectively, in these two rye Phaeosphaeria sp. isolates. Disease severity on the fifth leaf (GS15) of Polish wheat (Alba, Begra, and Liwilla), triticale (Bogo and Pinokio), and rye (Zduno) cultivars was assessed with one (resistant) to nine (susceptible) scales 14 days after inoculation. Aggressiveness of wheat-biotype P. nodorum isolate Sn26-1 and rye Phaeosphaeria sp. isolate Sn48-1 was significant (P < 0.01) in five cultivars except in the moderately resistant wheat cv. Liwilla. The rye Phaeosphaeria sp. isolate Sn48-1 severely affected Polish rye Zduno (8.3) and two triticale cultivars (6.5), while the infection by isolate Sn26-1 was moderate (3–4). On the contrary, the wheat-biotype P. nodorum isolate Sn26-1 was more aggressive on wheat (4.1 on moderately resistant Alba and 6.2 on highly susceptible Begra) than the rye Phaeosphaeria sp. isolate Sn48-1, which had a scale of 2.2 and 4.3, respectively. Under laboratory conditions, the rye isolate Sn48-1 was able to cross with the wheat-biotype P. nodorum isolate Sn26-1 that has an opposite mating-type (MAT1-2) gene, but few viable ascospores were produced (1). References: (1) P. C. Czembor and E. Arseniuk. Mycol. Res. 104:919, 2000. (2) A. Malkus et al. FEMS (Fed. Eur. Microbiol. Soc.) Lett. 249:49, 2005. (3) E. Reszka et al. Can. J. Bot. 83:1001, 2005. (4) M. J. Richardson and M. Noble. Plant Pathol. 19:159, 1970.

Unusual Antigenic and Genetic Characteristics of Human Respiratory Syncytial Viruses Isolated in Cuba

ABSTRACT The G protein of 23 strains of human respiratory syncytial virus isolated in Havana, Cuba, between October 1994 and January 1995 was analyzed at the antigenic and genetic level. All viruses reacted with 10 of 11 antibodies specific for the Long strain. Moreover, the G protein gene of the Cuban isolates had only five nucleotide differences from the sequence of the Long gene. The homogeneity of the Cuban isolates and their resemblance to an ancient strain, such as Long, are at odds with previous findings for viruses isolated in countries with a temperate climate and different socioeconomic status. The G proteins of three of four other viruses isolated in Havana 2 years later (1996) were also identical to those of the 1994-to-1995 isolates, and the fourth virus had a single extra nucleotide difference. This, again, is unusual, since no identical viruses had been isolated in different epidemics previously. The singular characteristics of the Cuban isolates reported here are discussed in terms of the epidemiological, climatic, and socioeconomic characteristics of Cuba.

Codon recognition during frameshift suppression in Saccharomyces cerevisiae.

A genetic approach has been used to establish the molecular basis of 4-base codon recognition by frameshift suppressor tRNA containing an extra nucleotide in the anticodon. We have isolated all possible base substitution mutations at the position 4 (N) in the 3'-CCCN-5' anticodon of a Saccharomyces cerevisiae frameshift suppressor glycine tRNA encoded by the SUF16 gene. Base substitutions at +1 frameshift sites in the his4 gene have also been obtained such that all possible 4-base 5'-GGGN-3' codons have been identified. By testing for suppression in different strains that collectively represent all 16 possible combinations of position 4 nucleotides, we show that frameshift suppression does not require position 4 base pairing. Nonetheless, position 4 interactions influence the efficiency of suppression. Our results suggest a model in which 4-base translocation of mRNA on the ribosome is directed primarily by the number of nucleotides in the anticodon loop, whereas the resulting efficiency of suppression is dependent on the nature of position 4 nucleotides.