Mapping RNA exit channel on transcribing RNA polymerase II by FRET analysis.

A simple genetic tag-based labeling method that permits specific attachment of a fluorescence probe near the C terminus of virtually any subunit of a protein complex is implemented. Its immediate application to yeast RNA polymerase II (pol II) enables us to test various hypotheses of RNA exit channel by using fluorescence resonance energy transfer (FRET) analysis. The donor dye is labeled on a site near subunit Rpb3 or Rpb4, and the acceptor dye is attached to the 5' end of RNA transcript in the pol II elongation complex. Both in-gel and single-molecule FRET analysis show that the growing RNA is leading toward Rpb4, not Rpb3, supporting the notion that RNA exits through the proposed channel 1. Distance constraints derived from our FRET results, in conjunction with triangulation, reveal the exit track of RNA transcript on core pol II by identifying amino acids in the vicinity of the 5' end of RNA and show that the extending RNA forms contacts with the Rpb7 subunit. The significance of RNA exit route in promoter escape and that in cotranscriptional mRNA processing is discussed.

Zernike phase plate cryoelectron microscopy facilitates single particle analysis of unstained asymmetric protein complexes.

Single particle reconstruction from cryoelectron microscopy images, though emerging as a powerful means in structural biology, is faced with challenges as applied to asymmetric proteins smaller than megadaltons due to low contrast. Zernike phase plate can improve the contrast by restoring the microscope contrast transfer function. Here, by exploiting simulated Zernike and conventional defocused cryoelectron microscope images with noise characteristics comparable to those of experimental data, we quantified the efficiencies of the steps in single particle analysis of ice-embedded RNA polymerase II (500 kDa), transferrin receptor complex (290 kDa), and T7 RNA polymerase lysozyme (100 kDa). Our results show Zernike phase plate imaging is more effective as to particle identification and also sorting of orientations, conformations, and compositions. Moreover, our analysis on image alignment indicates that Zernike phase plate can, in principle, reduce the number of particles required to attain near atomic resolution by 10-100 fold for proteins between 100 kDa and 500 kDa.