For virology class Suppose you were studying retroviral reverse transcription, a

Question

For virology class Suppose you were studying retroviral reverse transcription, and you were interested in developing an in vitro system (test-tube enzymatic reaction in the absence of cells) for studying this process. You purify reverse transcriptase and its associated RNase H activity, and prepare total RNA consisting of all the RNA present in retrovirus virions. You then set up an in vitro reaction with these components, and dNTPs (deoxynucleotide triphosphates) in an appropriate buffer for reverse transcription. Describe the nucleic acid product would you expect reverse transcriptase to produce in this situation. Would this system produce a double stranded DNA with duplicated LTRs? Explain why or why not.

Explanation / Answer

Retroviral Reverse transcriptase has three biochemical activities.

1. RNA dependent DNA polymerase activity

2. Ribonuclease H activity

3. DNA dependent DNA polymerase activity

These three sequential activities of Reverse transcriptase convert genomic RNA into double stranded cDNA which can be inserted into the host genome.

RNA dependent DNA polymerase activity produces cDNA strand which is termed as negative strand.

RNase H activity cleaves the RNA by an endonucleolytic mechanism in a DNA: RNA duplex.

DNA dependent DNA polymerase activity produces second strand of DNA. This strand is referred to as positive strand.

So, if you provide a reverse transcriptase with RNase H activity, it would generate a dsDNA product with duplicated LTRs.

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