芹菜(Apium graveolens L.)叶细胞质5SrRNA的序列测定

应用Gupta等和Tanaka等建立的RNA序列双向直读技术,并辅以部分酶解法、化学法等,测定了芹菜叶细胞质的5SrRNA的全序列:与菠菜和蕃茄细胞质已知5SrRNA序列进行了比较,发现它们之间在序列上有高度的保守性。     

The Helicase-Like Domain of Plant Potexvirus Replicase Participates in Formation of RNA 5′ Cap Structure by Exhibiting RNA 5′-Triphosphatase Activity

Open reading frame 1 (ORF1) of potexviruses encodes a viral replicase comprising three functional domains: a capping enzyme at the N terminus, a putative helicase in the middle, and a polymerase at the C terminus. To verify the enzymatic activities associated with the putative helicase domain, the corresponding cDNA fragment from bamboo mosaic virus (BaMV) was cloned into vector pET32 and the protein was expressed in Escherichia coli and purified by metal affinity chromatography. An activity assay confirmed that the putative helicase domain has nucleoside triphosphatase activity. We found that it also possesses an RNA 5'-triphosphatase activity that specifically removes the gamma phosphate from the 5' end of RNA. Both enzymatic activities were abolished by the mutation of the nucleoside triphosphate-binding motif (GKS), suggesting that they have a common catalytic site. A typical m(7)GpppG cap structure was formed at the 5' end of the RNA substrate when the substrate was treated sequentially with the putative helicase domain and the N-terminal capping enzyme, indicating that the putative helicase domain is truly involved in the process of cap formation by exhibiting its RNA 5'-triphosphatase activity.     

Blocked 5′ Termini in Brome Mosaic Virus RNA

All four components of brome mosaic virus RNA have m(7)G(5') ppp (5')Gp as their 5' terminus. The m(7)G can be removed by beta-elimination, resulting in the conversion to pppGp.     

Specific Interaction between the Hepatitis C Virus NS5B RNA Polymerase and the 3′ End of the Viral RNA

Hepatitis C virus (HCV) NS5B protein is the viral RNA-dependent RNA polymerase capable of directing RNA synthesis. In this study, an electrophoretic mobility shift assay demonstrated the interaction between a partially purified recombinant NS5B protein and a 3' viral genomic RNA with or without the conserved 98-nucleotide tail. The NS5B-RNA complexes were specifically competed away by the unlabeled homologous RNA but not by the viral 5' noncoding region and very poorly by the 3' conserved 98-nucleotide tail. A 3' coding region with conserved stem-loop structures rather than the 3' noncoding region of the HCV genome is critical for the specific binding of NS5B. Nevertheless, no direct interaction between the 3' coding region and the HCV NS5A protein was detected. Furthermore, two independent RNA-binding domains (RBDs) of NS5B were identified, RBD1, from amino acid residues 83 to 194, and RBD2, from residues 196 to 298. Interestingly, the conserved motifs of RNA-dependent RNA polymerase for putative RNA binding (220-DxxxxD-225) and template/primer position (282-S/TGxxxTxxxNS/T-292) are present in the RBD2. Nevertheless, the RNA-binding activity of RBD2 was abolished when it was linked to the carboxy-terminal half of the NS5B. These results provide some clues to understanding the initiation of HCV replication.     

Recent insights into noncanonical 5′ capping and decapping of RNA

The 5′ N⁷-methylguanosine cap is a critical modification for mRNAs and many other RNAs in eukaryotic cells. Recent studies have uncovered an RNA 5′ capping quality surveillance mechanism, with DXO/Rai1 decapping enzymes removing incomplete caps and enabling the degradation of the RNAs, in a process we also refer to as “no-cap decay.” It has also been discovered recently that RNAs in eukaryotes, bacteria, and archaea can have noncanonical caps (NCCs), which are mostly derived from metabolites and cofactors such as NAD, FAD, dephospho-CoA, UDP-glucose, UDP-N-acetylglucosamine, and dinucleotide polyphosphates. These NCCs can affect RNA stability, mitochondrial functions, and possibly mRNA translation. The DXO/Rai1 enzymes and selected Nudix (nucleotide diphosphate linked to X) hydrolases have been shown to remove NCCs from RNAs through their deNADding, deFADding, deCoAping, and related activities, permitting the degradation of the RNAs. In this review, we summarize the recent discoveries made in this exciting new area of RNA biology.     

Light-directed 5′→3′ synthesis of complex oligonucleotide microarrays

Light-directed synthesis of high-density microarrays is currently performed in the 3′→5′ direction due to constraints in existing synthesis chemistry. This results in the probes being unavailable for many common types of enzymatic modification. Arrays that are synthesized in the 5′→3′ direction could be utilized to perform parallel genotyping and resequencing directly on the array surface, dramatically increasing the throughput and reducing the cost relative to existing techniques. In this report we demonstrate the use of photoprotected phosphoramidite monomers for light-directed array synthesis in the 5′→3′ direction, using maskless array synthesis technology. These arrays have a dynamic range of >2.5 orders of magnitude, sensitivity below 1 pM and a coefficient of variance of <10% across the array surface. Arrays containing >150 000 probe sequences were hybridized to labeled mouse cRNA producing highly concordant data (average R² = 0.998). We have also shown that the 3′ ends of array probes are available for sequence-specific primer extension and ligation reactions.     

Uncoupling of Protein and Ribonucleic Acid Synthesis by 5′,5′,5′-Trifluoroleucine in Salmonella typhimurium

The addition of 5',5',5'-trifluoroleucine (fluoroleucine) to leucine auxotrophs of Salmonella typhimurium permitted protein but not ribonucleic acid (RNA) synthesis to continue after leucine depletion. The uncoupling of the formation of these macromolecules by fluoroleucine was apparent if RNA and protein synthesis was measured either by the uptake of radioactive precursors or by direct chemical determinations. The analogue did not appear to be an inhibitor of RNA formation, since it was as effective as leucine in permitting RNA synthesis in a leucine auxotroph upon the addition of small amounts of chloramphenicol. In contrast to these data, fluoroleucine allowed continued protein and RNA formation in a leucine auxotroph of Escherichia coli strain W. In addition, contrary to the results obtained with S. typhimurium, the analogue replaced leucine for repression of the leucine bio-synthetic enzymes as well as the isoleucine-valine enzymes. We propose that these ambivalent effects of fluoroleucine on repression and RNA and protein synthesis in the two strains are due to differences in the ability of the analogue to attach to the various species of leucine transfer RNA.     

Effect of nucleoside 5′-di- and 5′-tri-phosphates on pancreatic ribonuclease activity

1. ADP, ATP and GDP inhibited the phosphotransferase activity, the release of cyclic nucleotides from RNA, of ribonuclease. No significant inhibition was elicited by pyrimidine 5'-nucleoside diphosphates, CDP and UDP. 2. Inhibition by ADP, AMP, adenosine, adenine, NAD and NADP was insignificant at the concentrations tested. Small inhibition was observed with high concentrations of AMP and only when soluble RNA was the substrate. 3. Inhibition by ADP was found to be ;uncompetitive'. 4. Results seem to indicate that at least for optimum inhibition the polyphosphate of the purine nucleoside is essential. They further suggest that the inhibitor acts by combining with the enzyme only when the enzyme is bound to the substrate.     

5-Bromouridylyl-(3′ → 5′)-Adenosine Isolated from 5-Bromouracil-Induced Filaments of Escherichia coli

A dinucleoside monophosphate was isolated from 5-bromouracil-induced filaments of a thymine auxotroph of Escherichia coli K-12. The dinucleoside monophosphate was fractioned from a [(14)C]5-bromouracil-labeled perchloric acid extract using Dowex-1-formate ion-exchange chromatography. Sephadex chromatography revealed its molecular weight to be 710. Snake venom phosphodiesterase digest of the dinucleoside monophosphate yielded [(14)C]5-bromouridine and adenosine 5'-monophosphate. The presence of [(14)C]5-bromouracil in bacterial ribonucleic acid indicates that ribonucleic acid, which had incorporated 5-bromouracil, was the probable source of this dinucleoside monophosphate, 5-bromouridylyl-(3' --> 5')-adenosine.     

NrnA is a 5′-3′ exonuclease that processes short RNA substrates in vivo and in vitro

Bacterial RNases process RNAs until only short oligomers (2–5 nucleotides) remain, which are then processed by one or more specialized enzymes until only nucleoside monophosphates remain. Oligoribonuclease (Orn) is an essential enzyme that acts in this capacity. However, many bacteria do not encode for Orn and instead encode for NanoRNase A (NrnA). Yet, the catalytic mechanism, cellular roles and physiologically relevant substrates have not been fully resolved for NrnA proteins. We herein utilized a common set of reaction assays to directly compare substrate preferences exhibited by NrnA-like proteins from Bacillus subtilis, Enterococcus faecalis, Streptococcus pyogenes and Mycobacterium tuberculosis. While the M. tuberculosis protein specifically cleaved cyclic di-adenosine monophosphate, the B. subtilis, E. faecalis and S. pyogenes NrnA-like proteins uniformly exhibited striking preference for short RNAs between 2–4 nucleotides in length, all of which were processed from their 5′ terminus. Correspondingly, deletion of B. subtilis nrnA led to accumulation of RNAs between 2 and 4 nucleotides in length in cellular extracts. Together, these data suggest that many Firmicutes NrnA-like proteins are likely to resemble B. subtilis NrnA to act as a housekeeping enzyme for processing of RNAs between 2 and 4 nucleotides in length.     

Aminonucleosides and their derivatives. IVI Synthesis of the 3′-amino-3′-deoxynucleoside 5′-phosphates

A new procedure has been developed for the synthesis of 3′-amino-3′-deoxyribonucleosides of adenine, cytosine and uracil by condensing the trimethylsilylated bases with peracylated 3-azido-3-deoxyribose derivative. The azido group could subsequently be reduced to amino. The 5′-phosphates of these nucleosides have been prepared and the analogues have been tested for their ability to stimulate the ribosome-catalyzed reaction of 3′(2′)-O-(N-formylmethionyl)adenosine 5′-phosphate with phenylalanyl-tRNA.     

A Short Sequence within Two Purine-Rich Enhancers Determines 5′ Splice Site Specificity

Purine-rich enhancers are exon sequences that promote inclusion of alternative exons, usually via activation of weak upstream 3′ splice sites. A recently described purine-rich enhancer from the caldesmon gene has an additional activity by which it directs selection of competing 5′ splice sites within an alternative exon. In this study, we have compared the caldesmon enhancer with another purine-rich enhancer from the chicken cardiac troponin T (cTNT) gene for the ability to regulate flanking splice sites. Although similar in sequence and length, the two enhancers demonstrated strikingly different specificities towards 5′ splice site choice when placed between competing 5′ splice sites in an internal exon. The 32-nucleotide caldesmon enhancer caused effective usage of the exon-internal 5′ splice site, whereas the 30-nucleotide cTNT enhancer caused effective usage of the exon-terminal 5′ splice site. Both enhancer-mediated splicing pathways represented modulation of the default pathway in which both 5′ splice sites were utilized. Each enhancer is multipartite, consisting of two purine-rich sequences of a simple (GAR)_n repeat interdigitated with two enhancer-specific sequences. The entire enhancer was necessary for maximal splice site selectivity; however, a 5- to 7-nucleotide region from the 3′ end of each enhancer dictated splice site selectivity. Mutations that interchanged this short region of the two enhancers switched specificity. The portion of the cTNT enhancer determinative for 5′ splice site selectivity was different than that shown to be maximally important for activation of a 3′ splice site, suggesting that enhancer environment can have a major impact on activity. These results are the first indication that individual purine-rich enhancers can differentiate between flanking splice sites. Furthermore, localization of the specificity of splice site choice to a short region within both enhancers indicates that subtle differences in enhancer sequence can have profound effects on the splicing pathway.     

Effects of Adenosine 3′,5′-Monophosphate and Adenosine 5′-Monophosphate on Glycogen Degradation and Synthesis in Dictyostelium discoideum

Data are presented demonstrating that the presence in vivo of adenosine 3',5'-monophosphate (3',5'-AMP) causes a rapid depletion of glycogen storage material in the cellular slime mold. The effect of adenosine 5'-monophosphate (5'-AMP) is twofold, stimulating both glycogen degradation and synthesis. In pseudoplasmodia, cell-free extracts appear to contain at least two species of glycogen phosphorylase, one of which is severely inhibited by glucose-1-phosphate and another which is only partially inhibited by this hexose-phosphate. In some cases, 5'-AMP partially overcomes the inhibition by glucose-1-phosphate. Data presented here also indicate the existence of two forms of glycogen synthetase, the total activity of which does not change during 10 hr of differentiation from aggregation to culmination. During this period there is a quantitative conversion of glucose-6-phosphate-independent enzyme activity to glucose-6-phosphate-dependent activity. It is suggested that one effect of 3',5'-AMP is closely related to enzymatic processes involved in the rapid conversion of glycogen to cell wall material and other end products accumulating during sorocarp construction.