Investigation of Downstream DNA Effects on Elongational Pausing by E.

coli RNA Polymerase

Chandana Lanka*, Reema Khanchandani*, Cassidy Crook* # Judith R. Levin* Departments of *Biology and #Chemistry, Goucher College, 1021 Dulaney Valley Rd., Baltimore, MD 21204

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

Background and Experimental Approach

During transcription elongation, RNAP does not move smoothly along the DNA template strand as many may imagine but instead pauses at specific places (reviewed in Landick 2006). It is beneficial that the mechanisms of pausing be better understood as they serve as an important level of regulation during transcription elongation. Previous research has examined the influences of DNA and RNA sequence and secondary structure of the RNA transcript on pausing (Artsimovich & Landick 2000). Many pause sites have been shown to be influenced by DNA sequences downstream of the polymerase active site, however, the mechanism by which these downstream sequences affect pausing is not known. We are engaged in a systematic study of the effects of downstream DNA sequence and structure on elongation. Our experiments use techniques developed in previous in vitro transcription elongation experiments in which the transcriptional elongation complex (TEC) was reconstituted onto double-stranded DNA templates (oligonucleotides) to create scaffolds (Kyzer et al 2007). A short RNA primer is allowed to base pair to the DNA template strand oligonucleotide as the temperature is slowly reduced. When RNAP is added it binds to the DNA/RNA hybrid, and when a nontemplate strand oligonucleotide is added, and a transcription bubble forms. These complexes have been shown to have properties identical to those of promoterinitiated TECs. This system allows for the initiation step to be bypassed as no promoter is needed; furthermore, it allows for the easy manipulation of template sequences by the synthesis of new oligonucleotides. For transcription experiments, RNA was 5 end-labeled and was used to make TECs as described above (sequences of RNA and DNA oligonucleotides are shown with each experiment). TECs were allowed to elongate in the presence of all four NTPs (10 uM each) at 37C. Samples were taken at 15", 30", 1', 2', and 5'. A high concentration of all 4 NTPs (500 uM each) was then added and a final "chase" sample was taken after an additional 5' of elongation. Samples were analyzed on a 15% acrylamide/7M urea gel in 0.5X TBE and visualized by phosphorimaging. ImageQuant software used to quantify band intensities and pausing kinetics were analyzed according to Landick et al (1996). Another way to negate A-tract-induced bending is to substitute a G residue at the central position of the A-tract (Diekmann et al 1987). This is a less radical sequence change than inserting 4 Ts upstream, and also does not involve a sequence change at the actual site of the pause. Disrupting the 3 A-tracts downstream of the first pause reduced the half-life of that pause ~2-fold, and disruption of the 1st downstream A-tract created an equal effect, suggesting that only the 1st downstream A-tract is important in modulating the pause.

Preliminary Studies Conclusions

Pausing by E.coli RNA polymerase can be modulated by downstream sequences in a way that correlates with bending in these downstream sequences. A bent A5-tract located at positions +3 to +7 relative to a pause increases the half-life of that pause 2fold relative to a template containing the unbent sequence AAGAA at the same position.

Structure Vs. Sequence

Truncated Templates

Sequence swapping experiments helped elucidate the importance of sequences in the vicinity of the pause sites

A5N5wt

A5N5swap

T4A5Nwt

T4A5Nswap

swapped area

References and Acknowledgments :

Artsimovich, I. & Landick, R. (2000). Pausing by Bacterial RNA Polymerase is Mediated by Mechanistically Distinct Classes of Signals. PNAS. 97, 7090-7095. Landick, R. (2006). The Regulatory Roles and Mechanism of Transcriptional Pausing. Biochemical Society Transactions. Vol.34, Part 6. J. Gowrishankar & R. Harinarayanan. (2004). Why is transcription coupled to translation in bacteria? Molecular Microbiology. 54(3), 598603 Levin, J.R. & Chamberlin, M.J. (1987). Mapping and Characterization of Transcriptional Pause Sites in the Early Genetic Region of Bacteriophage T7. J. Mol. Biol. 196, 61-84. Diekmann et al. (1987) PNAS 84, 8257-8261. Kyzer, S., Ha, K.S., Landick, R., Palangat, M. (2007). Direct versus Limited-step Reconstitution Reveals Key Features of an RNA Hairpin-stabilized Paused Transcription Complex. Journal of Biochemistry. Vol. 282, 19020-19028. Walker J.D, Baker T.A., Bell S.P., Gann A., Levine M., Losick R. (2004). Molecular Biology of the Gene. 5th ed. Pearson Education Inc., San Francisco, CA. Vassylyer, D.G et al (2007) Natural 488, 157 - 162 Levin, J. R., Chamberlain, M. J. (1984) Mapping and Characterization of Transcription Pause Sites in the Early Genetic Region of Bacteriophage T7. J. Mol. Bio. 196, 61-64

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.