Biomedical subjects

Alla Shundrovsky

Publications and source records attributed to Alla Shundrovsky.

4 recordsLinked to original sources

PubMed2006

Probing SWI/SNF remodeling of the nucleosome by unzipping single DNA molecules.

Chromatin-remodeling enzymes can overcome strong histone-DNA interactions within the nucleosome to regulate access of DNA-binding factors to the genetic code. By unzipping individual DNA duplexes, each containing a uniquely positioned nucleosome flanked by long segments of DNA, we directly probed histone-DNA interactions. The resulting disruption-force signatures were characteristic of the types and locations of interactions and allowed measurement of the positions of nucleosomes with 2.6-base-pair (bp) precision. Nucleosomes remodeled by yeast SWI/SNF were moved bidirectionally along the DNA, resulting in a continuous position distribution. The characteristic distance of motion was approximately 28 bp per remodeling event, and each event occurred with a catalytic efficiency of 0.4 min(-1) per nM SWI/SNF. Remodeled nucleosomes had essentially identical disruption signatures to those of unremodeled nucleosomes, indicating that their overall structure remained canonical. These results impose substantial constraints on the mechanism of SWI/SNF remodeling.

Chromosomal Proteins, Non-Histone↗

PubMed2004

Sequence-dependent kinetic model for transcription elongation by RNA polymerase.

We present a kinetic model for the sequence-dependent motion of RNA polymerase (RNAP) during transcription elongation. For each NTP incorporation, RNAP has a net forward translocation of one base-pair along the DNA template. However, this process may involve the exploration of back-tracked and forward-tracked translocation modes. In our model, the kinetic rates for the reaction pathway, calculated based on the stabilities of the transcription elongation complex (TEC), necessarily lead to sequence-dependent NTP incorporation rates. Simulated RNAP elongation kinetics is in good agreement with data from transcription gels and single-molecule studies. The model provides a kinetic explanation for well-known back-tracked pauses at transcript positions with unstable TECs. It also predicts a new type of pause caused by an energetically unfavorable transition from pre to post-translocation modes.

Base Pairing↗

PubMed2004

A single-molecule technique to study sequence-dependent transcription pausing.

We present a technique that allows sequence-dependent analysis of transcription elongation using single-molecule optical trapping techniques. Observation of individual molecules of RNA polymerase (RNAP) allows determination of elongation kinetics that are difficult or impossible to accurately obtain from bulk studies, and provides high temporal resolution of the RNAP motion under a calibrated mechanical load. One limitation of previous single molecule studies was the difficulty in correlating the observed motion of RNAP with its actual position on the DNA template to better than approximately 100 bp. In this work, we improved the spatial precision of optical trapping studies of transcription to approximately 5 bp by using runoff transcription as an unambiguous marker of RNAP template position. This runoff method was sufficient to unequivocally locate and study a single known pause sequence (DeltatR2). By applying various loads to assist RNAP forward translocation, we specifically investigated elongation kinetics within this pause region and found that the dwell time at the pause sequence decreased with increasing assisting load. This observation is consistent with bulk biochemical studies that suggest RNAP reverse translocates, or "backtracks," at the DeltatR2 pause sequence.

Base Sequence↗

PubMed2002

Probing protein-DNA interactions by unzipping a single DNA double helix.

We present unzipping force analysis of protein association (UFAPA) as a novel and versatile method for detection of the position and dynamic nature of protein-DNA interactions. A single DNA double helix was unzipped in the presence of DNA-binding proteins using a feedback-enhanced optical trap. When the unzipping fork in a DNA reached a bound protein molecule we observed a dramatic increase in the tension in the DNA, followed by a sudden tension reduction. Analysis of the unzipping force throughout an unbinding "event" revealed information about the spatial location and dynamic nature of the protein-DNA complex. The capacity of UFAPA to spatially locate protein-DNA interactions is demonstrated by noncatalytic restriction mapping on a 4-kb DNA with three restriction enzymes (BsoBI, XhoI, and EcoRI). A restriction map for a given restriction enzyme was generated with an accuracy of approximately 25 bp. UFAPA also allows direct determination of the site-specific equilibrium association constant (K(A)) for a DNA-binding protein. This capability is demonstrated by measuring the cation concentration dependence of K(A) for EcoRI binding. The measured values are in good agreement with previous measurements of K(A) over an intermediate range of cation concentration. These results demonstrate the potential utility of UFAPA for future studies of site-specific protein-DNA interactions.

Base Sequence↗