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Abstract
Our research focuses on characterizing the nature and mechanisms of effects of downstream DNA on transcriptional pausing by E. coli RNA polymerase. One area of study involves the effect of A-tract-induced DNA bending on elongation. Using transcription elongation complexes (TECs) reconstituted in vitro on oligonucleotide scaffolds, we showed that a template containing four phased A-tracts ((GGGCA5T)4), pausing was observed in the three inter-A-tract regions, at each case at the 2nd of the 3 G residues. While our preliminary studies suggest that pause strength is sensitive to structural changes (bent vs. straight) in the downstream DNA, there remains the possibility that the effect is sequence-specific, resulting from the substitution of a GC-basepair for an AT-basepair, and not related to the change in trajectory of the DNA duplex. To address this question, we are taking two approaches. First, we are transcribing templates in which the nontemplate strand is truncated beyond the pause site, to ask whether the effect of downstream DNA at this particular pause depends on its existence as a DNA duplex. Second, we are constructing templates containing the nucleotide analogs diaminopurine (n2R) and inosine (I), substituting an n2R-T pair for the central A-T pair in the first downstream A-tract, or an I-C pair for the G-C pair in the AAGAA-containing template. These substitutions create templates with identical template-strand sequence but different bending status than the original templates (AAn2RAA is not bent, while AAIAA is bent1), which should help resolve structure- vs. sequence-specific effects of this downstream region on pausing. In a related previous study2, we observed sequences other than A-tracts that are able to exert downstream effects on pausing. As there is no a priori reason to expect that these sequences create local anomalies in DNA duplex structure, we are interested in determining what features of these sequences are important for their effects. We are in the process of constructing oligonucleotide templates containing these sequences for TEC reconstitution, in order to recapitulate the previously observed effects and begin dissecting them.
These pauses are critically dependent on upstream sequences, but are modulated by downstream sequences in a way that suggests a role of DNA bending
Inversion of the 1st downstream A-tract has an even greater modulating effect on pausing and this effect is negated by disruption of the A-tract
Sequence swapping experiments helped elucidate the importance of sequences in the vicinity of the pause sites
A5N5wt
A5N5swap
T4A5Nwt
T4A5Nswap
swapped area
We thank Dr. Bob Landick of U.Wisconsin Madison for RNA Polymerase, helpful advice, and use of laboratory facilities during a sabbatical leave by JL; this research is funded by NIH Grant #1R15GM081860-01 to JL.