Studies of Structure-Function Relationships in Transcriptional Pausing by Escherichia Coli RNA Polymerase

Students: Julia Nguyen (Dept. of Biological Sciences) & Laura Ojeda (Dept. of Biological Sciences)

Faculty Adviser: Judy Levine (Dept. of Biological Sciences & Chemistry)

Abstract

During transcriptional elongation, RNA polymerase (RNAP) pauses temporarily at certain DNA template sequences before resuming synthesis of an RNA chain.  Pausing serves to modulate the rate of RNA chain completion, and to coordinate interaction of RNAP with regulatory proteins that can affect termination, splicing and translation; hence it is important to understand the mechanism of pausing. Previous studies by others suggest that pausing is triggered when interactions between DNA and the enzyme's leading edge ("downstream" of the active site where RNA nucleotides are being added) are disrupted, leading to a temporary opening of the clamped structure of the elongating complex.  In this study, the influence of DNA sequence and structure downstream of a pause site are being investigated. Elongation complexes of Escherichia coli RNAP are artificially assembled on nucleic acid scaffolds composed of synthetic DNA and RNA oligonucleotides, and kinetics of RNA chain elongation are observed by electrophoresis of radioactively labeled RNA at various times after addition of nucleotide substrates.  We observe differences in pause strength with changes as subtle as the addition or removal of a single amino group in the minor groove of the DNA 5 basepairs downstream of the pause site.  The effect of downstream DNA shape (modulated by A-tract sequences that induce DNA bending) as well as the effect of RNAP mutations in the vicinity of the downstream DNA, which might be expected to influence the network of protein-protein and protein-nucleic acid interactions that contribute to pausing, is also being investigated.

Acknowledgments:

This work was supported by Goucher College and NIH AREA grant #R15GM081860-01

 
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