fur Biochemie9 BayerischeA new modification of silver staining is presented which utilizes two chemical properties of thiosulfate: image enhancement by pretreatment of fixed gels, and formation of soluble silver complexes which prevents unspecific background staining during image development. This procedure provides high sensitivity for proteins, RNA and DNA in the nanogram range on a colorless, transparent background. The performance of this method is documented by staining one-and two-dimensional patterns of plant leaf proteins. Moreover, we achieved, for the first time, the detection of the non-structural, tobacco mosaic virus-specific 126 kDa protein directly in the onedimensional protein pattern of infected protoplasts by a staining procedure.Julius-Maximilians-Universitat, Wurzburg

Translational stop codon readthrough provides a regulatory mechanism of gene expression that is extensively utilised by positive-sense ssRNA viruses. The misreading of termination codons is achieved by a variety of naturally occurring suppressor tRNAs whose structure and function is the subject of this survey. All of the nonsense suppressors characterised to date (with the exception of selenocysteine tRNA) are normal cellular tRNAs that are primarily needed for reading their cognate sense codons. As a consequence, recognition of stop codons by natural suppressor tRNAs necessitates unconventional base pairings in anticodon-codon interactions. A number of intrinsic features of the suppressor tRNA contributes to the ability to read non-cognate codons. Apart from anticodon-codon affinity, the extent of base modifications within or 3' of the anticodon may up- or down-regulate the efficiency of suppression. In order to out-compete the polypeptide chain release factor an absolute prerequisite for the action of natural suppressor tRNAs is a suitable nucleotide context, preferentially at the 3' side of the suppressed stop codon. Three major types of viral readthrough sites, based on similar sequences neighbouring the leaky stop codon, can be defined. It is discussed that not only RNA viruses, but also the eukaryotic host organism might gain some profit from cellular suppressor tRNAs.

Eukaryotic tRNAs are synthesized in the nucleus and need to be exported to the cytoplasm where they function in translation. tRNA export is mediated by exportin-t, which binds tRNA directly and with high affinity. tRNAs are initially synthesized as precursor molecules. Maturation to functional tRNA takes place in the nucleus, precedes export, and includes trimming of the 59 and 39 ends, posttranscriptional addition of the 39 CCA end, nucleoside modifications, and in some cases splicing. Here we address the question of how tRNA maturation is coordinated with export and thus how cytoplasmic accumulation of inactive maturation intermediates is avoided. This could, in principle, be achieved by nuclear retention of immature tRNA or by selective export of the fully mature form. We show that exportin-t has a strong preference for tRNA with correctly processed 59 and 39 ends and nucleoside modification. tRNA recognition by exportin-t can thus be considered as a quality control mechanism for these maturation steps prior to tRNA export. Surprisingly however, exportin-t can efficiently bind unspliced tRNA and intron-containing tRNA is exported when the rate of splicing is slow. During characterization of the exportin-t /tRNA interaction we found that exportin-t recognizes features in the tRNA that are conserved between prokaryotic and eukaryotic tRNAs. Our data suggest that correct tRNA shape, the 59 and 39 terminal ends, and the TCC loop are critical for exportin-t binding.