The recent development of an efficient transformation method and shuttle vectors for Haloferax vokanii has set the stage for rapid progress in archaebacterial molecular biology. We describe a shuttle-expression vector that can be selected for and maintained in either H. vokanji or Escherichia coli and permits the expression of cloned genes in H. volcanii. The vector, pWL204, was constructed by incorporating an H. vokanii tRNALYS gene promoter into a derivative of the H. vokcanii-E. coli shuttle vector pWL102. The vector has been used to express a modified, intron-containing, H. mediterranei tRNATrP gene (tRNATrP-0167). Transcription from the tRNALYS gene promoter in vivo was detected by Northern (RNA) analysis with an oligonucleotide probe complementary to the unique intron sequence of tRNATrP-0167. Dependence of transcription on the tRNALYS promoter was demonstrated by the absence of transcription when the promoter sequence was deleted from the vector and by mapping the transcription initiation site by primer extension.
RNA sequences containing 2'-amino pyrimidines that bind with high-affinity to human thyroid stimulating hormone (hTSH) were isolated from a random sequence library by an in vitro selection-amplification procedure. A representative RNA ligand (T-15) has an equilibrium dissociation constant (Kd) of 2.5 nM for its interaction with hTSH and can discriminate between other members of the glycohormone family; no detectable binding was observed at low micromolar concentrations of hCG (human chorionic gonadotropin), while measured Kd values for the interactions with hLH (human leutinizing hormone) and hFSH (human follicle stimulating hormone) were > 1 microM and approximately 0.2 microM, respectively. The detection of hTSH in a dot blot assay with radiolabeled T-15 RNA was demonstrated.
An in vitro assay system has been developed for the Halobacterium volcanii tRNA intron endonuclease using in vitro generated precursor RNAs. A partially purified enzyme preparation is capable of precise and accurate excision of the intron from the halobacterial tRNA(Trp) precursor. The cleavage reaction produces products having 5' hydroxyl and 2',3' cyclic phosphate termini. Processing of precursor molecules containing deletions within the exon regions indicates that the halobacterial endonuclease does not require intact mature tRNA structure in the substrate; this is in contrast to the eukaryotic endonuclease enzyme that has an absolute requirement for these structures. The large halobacterial tRNA(Trp) intron does not appear to be a primary site for recognition by the endonuclease, however, its removal affects cleavage efficiency. Through a comparison of the structural and sequence features of the halobacterial substrates and the precursors of other archaebacterial intron-containing precursors, a common element is proposed for the recognition of substrates by intron endonuclease.
Expression of the yeast tRNAPro(UGG) gene in Haloferax volcanii resulted in the production of a single stable transcript that had not undergone intron processing or processing of 5' and 3' flanking sequences. Mutation of the exon-intron boundary region of this RNA to produce a precursor RNA with the preferred halobacterial consensus exon-intron boundary structure did not restore intron processing. Processing of 5' and 3' flanking sequences was restored when the acceptor stem U6-U67 pair was changed to A6-U67. The significance of these results in defining the recognition requirements of tRNA maturation enzymes in the halophilic domain Archaea is discussed.In vitro studies suggest that the archaeal and eukaryal tRNA intron endonucleases use different mechanisms to identify intron-containing precursor RNAs and their cleavage sites (2,3,9,11,12,16,17,19,20). To further explore the relationship between these enzyme systems we have utilized a Haloferax volcanii plasmid expression vector (13) to express the Saccharomyces cerevisiae intron-containing tRNAPro(UGG) (hereafter referred to as tRNAPro) gene in H. volcanii (Fig. 1A). The yeast tRNAPro gene was chosen since it encodes a precursor RNA that contains an intron located at the same relative position in the mature tRNA as halophilic pre-tRNAs (15) but lacks the preferred halobacterial exon-intron boundary structure. In vitro studies show that H. volcanii tRNA intron endonuclease requires that each exon-intron cleavage site be located in a 3-nucleotide bulge loop and that these loops be separated by 4 bp (20). While the intron-containing yeast tRNAPro RNA can assume a structure in which the two cleavage sites are located in loops separated by 4 bp, neither loop is the preferred 3-nucleotide bulge loop (Fig. 1A). By examining processing of the wild-type tRNAPro preRNA and a mutant, which has the preferred halophile exon-intron structure, it is possible to determine if exon-intron boundary structure alone is sufficient to direct cleavage in vivo. Expression of the nonhalophilic tRNA in these cells also provides a means to monitor 5' and 3' processing reactions by Northern (RNA) analysis, without interference from chromosomal encoded tRNAs.In this report we describe the processing in H. volcanii of RNAs arising from the wild-type yeast tRNAPro gene and a mutant tRNAPro gene in which the exon-intron boundary regions are converted to the preferred halophilic structure. We also describe how changing the U6-U67 pair of the yeast tRNAPro acceptor stem to a Watson-Crick pair, A6-U67, effects RNase
Chemical mutagenesis of a previously reported RNA Diels-Alderase (DA22) was followed by in vitro selection based on [4 + 2] catalysis. New mutated families of RNA Diels-Alderases closely related in sequence space were obtained. The mutated Diels-Alderases selected showed significant improvements in catalytic efficiency (k(cat)/K(m)) as compared to the original DA22. The improvement in catalytic activity was primarily due to a decrease in K(m), but modest increases in k(cat) were also observed. The increase in catalytic activity of these new Diels-Alderases was found not to negatively affect their dienophile specificity. Surprisingly, one of the most active Diels-Alderases (DAM 40), a subtle sequence mutant of DA22, was found to show a new metal dependence and could function with Ni(2+) as the only transition-metal ion. Truncation experiments of DA22 showed that the region shown to be hypervariable at the 3'-end of the structure could be deleted without a significant decrease in the relative rate of Diels-Alder catalysis.
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