No abstract

Unmodified tRNAs are powerful systems to study the effects of posttranscriptional modifications and site-directed mutations on both the structure and function of these ribonucleic acids. To define the general limitations of synthetic constructs as models for native tRNAs, it is necessary to elucidate the conformational states of unmodified tRNAs as a function of solution conditions. Here we report the conformational properties of unmodified yeast tRNAPhe as a function of ionic strength, [Mg2+], and temperature using a combination of spectroscopic measurements along with chemical and enzymatic probes. We find that in low [Na+] buffer at low temperature, native yeast tRNAPhe adopts tertiary structure in the absence of Mg2+. By contrast, tertiary folding of unmodified yeast tRNAPhe has an absolute requirement for Mg2+. Below the melting temperature of the cloverleaf, unmodified yeast tRNAPhe exists in a Mg2+-dependent equilibrium between secondary and tertiary structure. Taken together, our findings suggest that although the tertiary structures of tRNAs are broadly comparable, the intrinsic stability of the tertiary fold, the conformational properties of intermediate states, and the stability of intermediate states can differ significantly between tRNA sequences. Thus, the use of unmodified tRNAs as models for native constructs can have significant limitations. Broad conclusions regarding "tRNA folding" as a whole must be viewed cautiously, particularly in cases where structural changes occur, such as during protein synthesis.

1. The localization of DNAase activity in rye germ nuclei revealed 48% of the total nuclear activity in the nucleoplasm and 4 % in the chromatin.2. The endonuclease present in the nucleoplasm was purified more than 300-fold as compared with the specific activity in homogenized nuclei by means of ammonium sulphate fractionation and CM-cellulose and CM-Sephadex column chromatography. The preparation gave a single band on polyacrylamide gels. Its molecular weight is 45000, pH optimum 5.4. The preparation does not exhibit activity of non-specific phosphomonoesterase and phosphodiesterase.3 . The nuclease catalyses the hydrolysis of denatured DNA and RNA to acid-soluble 5'-phosphate-terminated products and cleaves the phosphomonoester linkage of 3'AMP.4. Synthetic polyribonucleotides are hydrolysed at a rate decreasing in the order : poly(U) The endonuclease shows a high preference for single-stranded nucleic acids. It does not depolymerise double-stranded poly(U) . poly(A).6. The nuclease attacks covalently closed circular bacteriophage PM2 DNA, plasmid ColEl and plasmid pBR322 DNA, converting it initially into the open-circular (relaxed) form and subsequently into the linear form. This reaction is strongly dependent on ionic strength.7. The enzyme nicks the supercoiled DNA molecule of phage PM2 in only one place. At high enzyme concentrations several nicks in such a molecule are observed.