Enzymatic incorporation of 2'-thio-CTP into the HDV ribozyme.
We have synthesized the analogue 2'-deoxy-2'-thio-CTP (CTP-SH) and tested its ability to support RNA transcription in place of CTP. The modified nucleotide in a transcription reaction and in the absence of CTP generated the appropriately sized fragment when a mutant T7 polymerase (Y639F) was used. Wild-type polymerase was unable to generate RNA under the same conditions. Transcription was optimal around pH 7.5 and was dependent upon CTP-SH concentration. Transcripts containing the analogue were efficiently isolated using a thiol-activated sepharose column. Insertion of CTP-SH into the HDV ribozyme, replacing all cytidine residues with 2'-thiocytidine, appears to inhibit self-cleaving activity, even in the presence of manganese. The ability to introduce the CTP-SH analogue enzymatically into RNA opens the way for new structure-function studies where the 2'-hydroxyl can be efficiently replaced by a thiol group.
Raines
K
K
Department of Biological Science, SUNY at Buffalo, New York 14260, USA.
Gottlieb
P A
PA
D002846:Chromatography, Affinity; D003570:Cytidine Triphosphate / Q000031:analogs & derivatives*; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism; D003845:Deoxycytosine Nucleotides / Q000138:chemical synthesis* / Q000378:metabolism; D003698:Hepatitis Delta Virus / Q000235:genetics*; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D016337:RNA, Catalytic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013873:Thionucleotides / Q000138:chemical synthesis* / Q000378:metabolism; D014158:Transcription, Genetic; D014764:Viral Proteins
C111174:2'-deoxy-2'-thiocytidylic acid; D003845:Deoxycytosine Nucleotides; D016337:RNA, Catalytic; D013873:Thionucleotides; D014764:Viral Proteins; D008345:Manganese; D003570:Cytidine Triphosphate; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D008274:Magnesium