Reiterative copying byE.coliRNA polymerase during transcription initiation of mutant pBR322 tet promoters

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Abstract
The major in vitro transcripts from the tet promoter of pBR322 derivatives pTA22 and pTA33 have heterogeneous 5'' ends consisting of variable lengths of oligo(A). Their structure is 5''pppAnU..., where n ranges from 1 to > 12, but the template strand can encode at most four A residues at the site of transcription initiation. The abundance of additional A residues at the 5'' end of the pTA22 and pTA33 tet transcripts could be reduced by elevating the concentration of UTP, but even at high concentrations (> 1 mM) non-cognate A residues were still observed. Aberrant initiation was not artifactual since the major and minor transcripts of the pBR322 tet promoter region, and other transcripts arising from minor promoters on pTA22 or pTA33 DNA all had unique 5'' termini. Mixing experiments showed that RNA polymerase did not utilize pppA2-4-OH produced by abortive initiation as primers. The data suggest that the initial nascent RNA chain ''slips'' in the 5'' direction during elongation opposite T4 on the template strand causing RNA polymerase to reiteratively add A residues to the 5'' end of the transcript. The generality and possible significance of this mechanism is discussed.