Revisiting the central dogma one molecule at a time

Abstract

The faithful relay and timely expression of genetic information depend on specialized molecular machines, many of which function as nucleic acid translocases. The emergence over the last decade of single-molecule fluorescence detection and manipulation techniques with nm and Å resolution and their application to the study of nucleic acid translocases are painting an increasingly sharp picture of the inner workings of these machines, the dynamics and coordination of their moving parts, their thermodynamic efficiency, and the nature of their transient intermediates. Here we present an overview of the main results arrived at by the application of single-molecule methods to the study of the main machines of the central dogma.

Figure 1. φ29 packaging motor

(A) Cryo-electron microscopy of the packaging motor. Left: Packaging motor with capsid and DNA modeled in for scale. Right: Close up on packaging motor. Modified from (Morais et al., 2008a). (B) Optical Tweezers Packaging Assay. Left: An optical trap exerts a force, F, on a single packaging bacteriophage while monitoring the length of the unpackaged DNA, L. Right: DNA length versus time. Different colors correspond to different concentrations of [ATP]. (C) High resolution packaging reveals a burst-dwell packaging mechanism. Left: Cartoon layout of high resolution packaging assay. Right: Schematic diagram of the kinetic events that occur during the dwell and burst phases overlaid on packaging data.

Figure 2. Single-molecule studies of helicases and mechanistic insights

(A) Single-molecule hairpin assay for NS3 helicase: cartoon representation of the experimental set up using optical tweezers to study translocation and unwinding of double stranded RNA by individual NS3 helicase; (B) representative real time unwinding trajectory of NS3 helicase on the hairpin substrate collected at 1 mM ATP, the burst of NS3 activity is noted by arrows, which has an average size of 11±3 bp. Bottom: Interactions between helicases and nucleic acid substrates. (C) Possible mode of binding in NS3 helicase. The binding of 3′ single strand is observed in cocrystal structures between NS3 and single-stranded nucleic acids. However, the binding of 5′ single strand has not been observed in any crystal structures but suggested from single-molecule studies. (D) Hexameric helicase, for example, T7 gp4 DNA helicase, extrudes one strand of the DNA through the center hole of the helicase while displacing the other strand.

Figure 3. Transcription through the nucleosome

(A). Hodges and Bintu follow Pol II transcription through the nucleosome in real time. They observe an increase in the probability of nucleosome passage with ionic strength, as well as an increase in pause density and pause duration in the vicinity of the nucleosome. Their model supports a passive mechanism of motion that depends on thermal fluctuations of the DNA-nucleosome interactions. (B). Jin et al. use an unzipping technique to infer the position of the polymerase after transcription has occurred. They also observe increased pausing within the nucleosomal sequence and verify nucleosome-induced polymerase backtracking of 10–15 bps. The inclusion of RNase or a trailing polymerase limits backtracking and increases the passage probability (Adapted from Hodges & Bintu et al. and Jin et al.).

Figure 4. Single-molecule studies of ribosomes

(A) Experimental design for monitoring single ribosome translation in real time. The ribosome was stalled at the 5′ side of the mRNA hairpin construct, which was then held between two polystyrene beads. Drawings are schematic and not to scale. (B) Single ribosome trajectory through an mRNA hairpin as in (A). Data obtained at constant force (lower panel). The arrows represent individual codon steps.