Reverse transcriptase and its associated ribonuclease H: interplay of two enzyme activities controls the yield of single-stranded cDNA

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Abstract
The synthesis of single-stranded globin cDNA [complementary DNA] by the RNA-directed DNA polymerase activity of [avian myeloblastosis virus] reverse transcriptase in the presence of oligothymidylate primers was investigated in order to determine the limitations to higher yields. The associated RNase H activity, an integral part of reverse transcriptase, plays a large role in the synthesis of the first strand of cDNA and the interplay of the 2 enzyme activities for any specific set of conditions determines the yield of single-stranded products. In both the presence and the absence of polymerization, the associated RNase H catalyzed the deadenylation of mRNA, producing molecules that were somewhat shorter, highly homogeneous in size, and fully translatable into globin protein. They were also entirely lacking in the ability to serve as templates for cDNA synthesis. The reaction was completely dependent on oligothymidylate and completely independent of deoxyribonucleoside triphosphates. The initial rate of deadenylation was one-fourth the initial rate of initiation of polymerization when saturating levels of deoxyribonucleoside triphosphates were used in the polymerase reaction. In the presence of RNase H activity, the DNA polymerase catalyzed the synthesis of an array of cDNA including some that were full length. The initiation of polymerization was rate limiting: once synthesis had begun, it required 1-1.5 min to transcribe globin mRNA. However, most primers that were elongated were aborted prematurely. Maximum synthesis of full-length cDNA required stoichiometric levels of enzyme and high triphosphate levels, but regardless of conditions, the sum of completed cDNA and deadenylated mRNA accounted for only 50% of the input mRNA. The data fit a model in which synthesis of full-length cDNA molecules depends on the arrangement of primers and transcription initiation complexes on the poly(A) tail of mRNA.

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