Nucleic Acids Research, 1990, Vol. 18, No. 12 3529-3535
© 1990
MOLECULAR BIOLOGY |
Transcriptional slippage occurs during elongation at runs of adenine or thymine in Escherichia coli
1Department of Human Genetics, 743 Wintrobe Building, University of Utah Salt Lake City, UT 84132 2Howard Hughes Medical Institute, 743 Wintrobe Building, University of Utah UT 84132 3Biology Department, Wilson Hall, University of North Carolina Chapel Hill, NC 27514, USA
*To whom correspondence should be addressed
Received March 16, 1990. Revised May 16, 1990. A run of 11 adenine or thymine residues at the 5' end of an out-of-frame lacZ gene causes a high level of ß-galactosidase expression in E. coli. This effect was not observed for a run of guanine residues. Reverse transcription of mRNA isolated from E. coli containing the run of 11 A's reveals heterogeneity of transcript length while reverse transcription of mRNA isolated from S.cerevisiae containing the same gene shows no heterogeneity. Protein sequencing of the ß-galactosidase molecules derived from the out-of-frame construct containing a run of adenines reveals the addition of a lysine at the run. A new method was developed where messages small enough to allow resolution of single nucleotide differences on an acrylamide gel are electrophoresed, electroblotted onto nylon and probed. This confirmed the reverse transcription results and showed that additional residues can be added to transcripts derived from DNA containing 10 or 11 thymine residues. A mechanism for slippage is discussed where the A-U rich RNA-DNA hybrid can denature during elongation and rehybridize in an offset position, causing the addition of extra residues to the transcript.
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