栝楼核糖核酸酶在RNA序列分析中的应用

栝楼核糖核酸酶（RNase TCS）对U碱基具有高度的专一性,在无脲、pH3.5、50℃时,它几乎都在-NP ↓ U-处裂解RNA.它与RNase T1,U₂和有限的碱水解一起,可用于直接的酶法RNA序列分析．     

A sensitive RNase protection assay using33P labeled antisense riboprobes

This article presents for the first time a modified protocol for RNase protection analysis that allows the substitution of³²P with³³P without loss of the high sensitivity of this method achieved with³²P. With this protocol, we were able to detect at least 1 pg of specific mRNA. In the RNase protection analysis³³P labeled riboprobes are more advantageous with regard to an easier handling and better resolution.     

Structure,mechanism and evolution of chloroplast transfer RNA processing systems

Chloroplasts of land plants have an active transfer RNA processing system, consisting of an RNase P-like 5 endonuclease, a 3 endonuclease, and a tRNA:CCA nucleotidyltransferase. The specificity of these enzymes resembles more that of their eukaryotic counterparts than that of their cyanobacterial predecessors. Most strikingly, chloroplast RNase P activity almost certainly resides in a protein, rather than in an RNA protein complex as in Bacteria, Archaea, and Eukarya. The chloroplast enzyme may have evolved from a preexisting chloroplast NADP-binding protein. Chloroplast RNase P cleaves pre-tRNA by a reaction mechanism in which at least one of the Mg²⁺ ions utilized by the bacterial ribozyme RNase P is replaced by an amino acid side chain.Abbreviations pre-tRNA precursor to tRNA - pCp cytidine 5, 3-bisphosphate - IC₅₀ inhibitor concentration giving 50% inhibition - GAPDH glyceraldehyde 3-phosphate dehydrogenase     

Changes occurring during aging and senescence in a submerged aquatic angiosperm (Potamogeton pectinatus)

Changes occurring during aging and senescence of leaves of a submerged aquatic angiosperm ( Potamogeton pectinatus L.) were studied. Total chlorophyll and chlorophylls a and b were maximal in mature, and minimal in old leaves. The chlorophyll a to b ratio was highest in mature leaves. During senescence, the chlorophyll content and the ratio of chlorophyll a to b decreased. The content of DNA, RNA, protein and dry weight, and the activity of alkaline pyrophosphatase decreased while free amino acids, the activity of protease, RNase and acid pyrophosphatase, and the ratio of acid to alkaline pyrophosphatase activity increased during aging and senescence. Kinetin (0.23 m M ) deferred leaf senescence by delaying the loss of chlorophyll, protein, nucleic acids and dry weight, and reducing the rise in free amino acids, the activity of protease, RNase and acid pyrophosphatase and the ratio of acid to alkaline pyrophosphatase activity; while both 0.69 m M ethrel and 0.075 m M ABA hastened senescence. Kinetin pretreatment for an optimum period (12 h) followed by ethrel or ABA treatment partially erased the senescence-promoting effect of the latter. But treatments in a reverse order markedly reduced the delaying effect of kinetin on senescence.     

Non-cognizable ribonucleotide, 2''AMP, binds to a mutant ribonuclease T1 (Y45W) at a new base-binding site but not at the guanine-recognition site.

Complex of a mutant ribonuclease T₁ (Y4SW) with a non-cognizable ribonucleotide, 2′AMP, has been determined and refined by X-ray diffraction at 1.7 Å resolution. The 2′AMP molecule locates at a new base-binding site which is remote from the guanine-recognition site, where 2′GMP was found to be bound. The nucleotide adopts the anti conformation of the glycosidic bond and C3′-exo sugar pucker. There exists a single hydrogen bond between the adenine base and the enzyme, and, therefore, the site found is apparently a non-specific binding site. The results indicate that the binding of 2′AMP to the guanine-recognition site is weaker than that to the new binding site.     

3D structure of bovine pancreatic ribonuclease A in aqueous solution: An approach to tertiary structure determination from a small basis of1H NMR NOE correlations

Summary A method is proposed to generate initial structures in cases where the distance geometry method may fail, such as when the set of¹H NMR NOE-based distance constraints is small in relation to the size of the protein. The method introduces an initial correlation between the and backbone angles (based on empirical observations) which is relaxed in later stages of the calculation. The obtained initial structures are refined by well-established methods of energy minimization and restrained molecular dynamics. The method is applied to determine the solution structure of Ribonuclease A (124 residues) from a NOE basis consisting of 467 NOE cross-correlations (97 intra-residue, 206 sequential, 23 medium-range and 141 long-range) obtained at 360 MHz. The global shape and backbone overall fold of the eight final refined structures are close to those shown by the crystal structure. A meaningful difference in the positioning of the catalytically important His¹¹⁹ side chain in the solution and crystal structures has been detected.