Single-molecule tracking of mRNA exiting from RNA polymerase II

Abstract

Single-pair fluorescence resonance energy transfer was used to track RNA exiting from RNA polymerase II (Pol II) in elongation complexes. Measuring the distance between the RNA 5' end and three known locations within the elongation complex allows us determine its position by means of triangulation. RNA leaves the polymerase active center cleft via the previously proposed exit tunnel and then disengages from the enzyme surface. When the RNA reaches lengths of 26 and 29 nt, its 5' end associates with Pol II at the base of the dock domain. Because the initiation factor TFIIB binds to the dock domain and exit tunnel, exiting RNA may prevent TFIIB reassociation during elongation. RNA further extends toward the linker connecting to the polymerase C-terminal repeat domain (CTD), which binds the 5'-capping enzyme and other RNA processing factors.

Fig. 1.

Pol II structure and labeling. (A) Schematic showing the oligonucleotides used in the single-molecule experiments for the formation of elongation complexes. Filled and open circles denote nucleotides whose positions are known and unknown, respectively, from crystallographic studies (3). Highlighted are the labeling positions on the template DNA and RNA. (B) Top view of the complete Pol II elongation complex structure (3), indicating labeling sites. The core polymerase is shown in surface representation (in gray), and the backbone of the template DNA (blue), nontemplate DNA (cyan), and RNA (red) and the heterodimer Rpb4/7 (Rpb4 in red and Rpb7 in blue) are displayed with cartoon diagrams. The positions that were used to attach dye molecules, that is, Rpb7, residues Rpb7–C94, and Rpb7–C150, as well as the template DNA at position −10 (DNA1) and +3 (DNA2), are shown in green. At Right is a close-up view of the labeling region highlighting the distances between Rpb7–C150 and DNA1 as well as Rpb7–C150 and DNA2.

Fig. 2.

sp-FRET time traces and histograms. Exemplary time traces and complete histograms for the FRET pairs DNA1–RNA17 (A and B) and DNA1–RNA26 (C and D). The time traces show the fluorescence intensities of the donor (green) and the acceptor (red) (thin lines correspond to the actual signal and thick lines correspond to a 10-point sliding average) as well as the computed FRET signal (blue). (A) The trajectory of the RNA17–DNA1 pair shows constant fluorescence intensities for the donor and acceptor molecules until bleaching of the acceptor after ≈12 s. At this point, the donor intensity increases and remains constant until the donor itself photo-bleaches after ≈22 s. The computed FRET efficiency is constant, except for small fluctuations attributable to photon-counting noise. (B) The histogram of 337 such sp-FRET trajectories shows a single peak, which can be fitted with a Gaussian distribution that is centered at E = 0.92. In contrast, the FRET efficiency in C for the DNA1–RNA26 sample is not constant but varies between E ≈ 0.85 and E ≈ 0.6. Accordingly the histogram in D of 224 such trajectories can be fitted with two Gaussian fits centered at E = 0.58 and E = 0.85.

Fig. 3.

Alternating laser excitation. The graph shows an exemplary time trace of the DNA1–RNA26 sample. The excitation laser was alternated frame by frame between 633 nm (direct–acceptor excitation, Upper) and 532 nm (FRET excitation, Lower) (34). Actual data and a 10-point sliding average are shown as thin and thick lines, respectively.

Fig. 4.

The position of the nascent RNA within the elongation complex as determined from sp-FRET measurements. (A) Back view of the elongation complex. The position of the dye molecule attached to the 5′ end is illustrated by a single sphere with a radius of 5 Å for RNA17 (orange), RNA20 (red), RNA23 (blue), and RNA29 (green) and by a pair of spheres for RNA26 (purple), indicating the presence of two states. (B) Cut-away view revealing the paths of the RNA on the interior and exterior. This figure was prepared using UCSF Chimera (46).