Identification of a complex associated with processing and polyadenylation in vitro of herpes simplex virus type 1 thymidine kinase precursor RNA.
Cleavage and polyadenylation of substrate RNAs containing the herpes simplex virus type 1 (HSV-1) thymidine kinase (tk) gene polyadenylation signal region were examined in HeLa cell nuclear extract. 3'-End RNA processing was accurate and efficient and required ATP and Mg2+. Cleavage, but not polyadenylation, occurred in the presence of EDTA or when ATP was replaced with 3' dATP (cordycepin) or AMP(CH2)PP, a nonhydrolyzable analog of ATP. Processing in vitro and in vivo showed the same signal element requirements: a series of substrates containing linker scanning, internal deletion, and small insertion mutations was processed with the same relative efficiencies and at the same sites in vitro and in vivo. A complex involved in 3'-end RNA processing was identified by gel mobility shift analysis. This complex formed rapidly, reached a maximum level after 20 to 30 min, and was much reduced after 2 h. Very little complex was formed at 0 degree C or with substrates lacking a polyadenylation signal. Entry of 32P-labeled tk substrate into the complex could be prevented by addition of excess 35S-labeled tk or adenovirus L3 precursor RNAs. Competition was not observed with tk RNAs lacking a complete polyadenylation signal.
Zhang
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Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03756.
Cole
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D000255:Adenosine Triphosphate / Q000378:metabolism; D002467:Cell Nucleus / Q000502:physiology; D002872:Chromosome Deletion; D004252:DNA Mutational Analysis; D004591:Electrophoresis, Polyacrylamide Gel; D005814:Genes, Viral; D006367:HeLa Cells; D007700:Kinetics; D009694:Nucleic Acid Precursors / Q000378:metabolism*; D011061:Poly A / Q000378:metabolism*; D012323:RNA Processing, Post-Transcriptional; D012333:RNA, Messenger / Q000378:metabolism*; D012261:Ribonucleoproteins / Q000502:physiology*; D018139:Simplexvirus / Q000235:genetics*; D013937:Thymidine Kinase / Q000235:genetics*
D009694:Nucleic Acid Precursors; D012333:RNA, Messenger; D012261:Ribonucleoproteins; D011061:Poly A; D000255:Adenosine Triphosphate; D013937:Thymidine Kinase