Reverse transcription and RNA second. structure

Terry Hanzlik terryh at ento.csiro.au
Tue Dec 13 03:22:59 EST 1994


Hello netters,
I recently came to grief with a deletion artifact of reverse transcription
while sequencing a cDNA of a tetravirus.  The murine moloney
lukemia RT and its derivative, Superscript I (BRL), predictably jumped
the same 50 bp region.  We were lucky that a frameshift occured
and could detect the deletion.  The enzymes jumped in the majority 
of cases and of 3 cDNA clones sequenced, all had the same deletion.
The deletion was ascertained only after sequencing the RNA
directly with two different RTs, one of the few "luxuries" for a 
RNA virus sequencer.  When sequencing the RNA directly, you could 
see the enzymes jumping the region on the sequence ladder.

The AMV and Superscript II (BRL) RTs did not jump  as well as the.
RT activities of Taq and Tth DNA pols.

The problem seem to be caused by very stable 50 bp stem-loop that had a
hexamer sequence directly repeated on both sides of its base.  We
figured the repeat sequence was important to the phenomenon and
not caused by just the secondary structure.  We postulated
that when the RT came to the 3' base of the stem, it paused having
synthesized cDNA to all or part of the repeat sequence on that
side of the loop structure.  The cDNA separated from the RNA 
then reannealed to its complementary sequence on the loop's 5' side
and continued along the RNA strand.  The RNAse H activity of
MuMLV RT and SS I aided the strand separation while the total
lack of RNAse H activity in SS II prevented this occuring for
that enzyme.  

A paper describing this phenomenon is in preparation.  We could
not find any other predictable jumping by RTs in the literature.

Thanks Brian for posting your experience and bringing it to our
attention. 

Regards,
Terry Hanzlik, terryh at ento.csiro.au
CSIRO Division of Entomology
Box 1700
Canberra, ACT  2601
Australia
 

> 
> Can reverse transcriptase jump over a loop of secondary structure and
> transcribe further, yelding shorter transcripts?
> Is there any published documentation of this phenomenon?
> 
> 




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