Harold D. Kim

tRNA selection and kinetic proofreading in translation

Using single-molecule methods we observed the stepwise movement of aminoacyl-tRNA (aa-tRNA) into the ribosome during selection and kinetic proofreading using single-molecule fluorescence resonance energy transfer (smFRET). Intermediate states in the pathway of tRNA delivery were observed using antibiotics and nonhydrolyzable GTP analogs. We identified three unambiguous FRET states corresponding to initial codon recognition, GTPase-activated and fully accommodated states. The antibiotic tetracycline blocks progression of aa-tRNA from the initial codon recognition state, whereas cleavage of the sarcin-ricin loop impedes progression from the GTPase-activated state. Our data support a model in which ribosomal recognition of correct codon-anticodon pairs drives rotational movement of the incoming complex of EF-Tu–GTP–aa-tRNA toward peptidyl-tRNA during selection on the ribosome. We propose a mechanistic model of initial selection and proofreading.

Mg2+-dependent conformational change of RNA studied by fluorescence correlation and FRET on immobilized single molecules

Fluorescence correlation spectroscopy (FCS) of fluorescence resonant energy transfer (FRET) on immobilized individual fluorophores was used to study the Mg2+-facilitated conformational change of an RNA three-helix junction, a structural element that initiates the folding of the 30S ribosomal subunit. Transitions of the RNA junction between open and folded conformations resulted in fluctuations in fluorescence by FRET. Fluorescence fluctuations occurring between two FRET states on the millisecond time scale were found to be dependent on Mg2+ and Na+ concentrations. Correlation functions of the fluctuations were used to determine transition rates between the two conformations as a function of Mg2+ or Na+ concentration. Both the opening and folding rates were found to vary with changing salt conditions. Assuming specific binding of divalent ions to RNA, the Mg2+ dependence of the observed rates cannot be explained by conformational change induced by Mg2+ binding/unbinding, but is consistent with a model in which the intrinsic conformational change of the RNA junction is altered by uptake of Mg2+ ion(s). This version of FCS/FRET on immobilized single molecules is demonstrated to be a powerful technique in the study of conformational dynamics of biomolecules over time scales ranging from microseconds to seconds.

Peptide bond formation destabilizes Shine–Dalgarno interaction on the ribosome

The ribosome is a molecular machine that translates the genetic code contained in the messenger RNA into an amino acid sequence through repetitive cycles of transfer RNA selection, peptide bond formation and translocation. Here we demonstrate an optical tweezer assay to measure the rupture force between a single ribosome complex and mRNA. The rupture force was compared between ribosome complexes assembled on an mRNA with and without a strong Shine–Dalgarno (SD) sequence—a sequence found just upstream of the coding region of bacterial mRNAs, involved in translation initiation. The removal of the SD sequence significantly reduced the rupture force in complexes carrying an aminoacyl tRNA, Phe-tRNAPhe, in the A site, indicating that the SD interactions contribute significantly to the stability of the ribosomal complex on the mRNA before peptide bond formation. In contrast, the presence of a peptidyl tRNA analogue, N-acetyl-Phe-tRNAPhe, in the A site, which mimicked the post-peptidyl transfer state, weakened the rupture force as compared to the complex with Phe-tRNAPhe, and the resultant force was the same for both the SD-containing and SD-deficient mRNAs. These results suggest that formation of the first peptide bond destabilizes the SD interaction, resulting in the weakening of the force with which the ribosome grips an mRNA. This might be an important requirement to facilitate movement of the ribosome along mRNA during the first translocation step.

Site-specific labeling of the ribosome for single-molecule spectroscopy.

Single-molecule fluorescence spectroscopy can reveal mechanistic and kinetic details that may not be observed in static structural and bulk biochemical studies of protein synthesis. One approach requires site-specific and stable attachment of fluorophores to the components of translation machinery. Fluorescent tagging of the ribosome is a prerequisite for the observation of dynamic changes in ribosomal conformation during translation using fluorescence methods. Modifications of the ribosomal particle are difficult due to its complexity and high degree of sequence and structural conservation. We have developed a general method to label specifically the prokaryotic ribosome by hybridization of fluorescent oligonucleotides to mutated ribosomal RNA. Functional, modified ribosomes can be purified as a homogenous population, and fluorescence can be monitored from labeled ribosomal complexes immobilized on a derivatized quartz surface.

The role of fluctuations in tRNA selection by the ribosome

The detailed mechanism of how the ribosome decodes protein sequence information with an abnormally high accuracy, after 40 years of study, remains elusive. A critical element in selecting correct transfer RNA (tRNA) transferring correct amino acid is “induced fit” between the ribosome and tRNA. By using single-molecule methods, the induced fit mechanism is shown to position favorably the correct tRNA after initial codon recognition. We provide evidence that this difference in positioning and thermal fluctuations constitutes the primary mechanism for the initial selection of tRNA. This work demonstrates thermal fluctuations playing a critical role in the substrate selection by an enzyme.

Probing the elastic limit of DNA bending

Sharp bending of double-stranded DNA (dsDNA) plays an essential role in genome structure and function. However, the elastic limit of dsDNA bending remains controversial. Here, we measured the opening rates of small dsDNA loops with contour lengths ranging between 40 and 200 bp using single-molecule Fluorescence Resonance Energy Transfer. The relationship of loop lifetime to loop size revealed a critical transition in bending stress. Above the critical loop size, the loop lifetime changed with loop size in a manner consistent with elastic bending stress, but below it, became less sensitive to loop size, indicative of softened dsDNA. The critical loop size increased from ∼60 bp to ∼100 bp with the addition of 5 mM magnesium. We show that our result is in quantitative agreement with the kinkable worm-like chain model, and furthermore, can reproduce previously reported looping probabilities of dsDNA over the range between 50 and 200 bp. Our findings shed new light on the energetics of sharply bent dsDNA.

Biofilm formation in geometries with different surface curvature and oxygen availability

Bacteria in the natural environment exist as interface-associated colonies known as biofilms . Complex mechanisms are often involved in biofilm formation and development. Despite the understanding of the molecular mechanisms involved in biofilm formation, it remains unclear how physical effects in standing cultures influence biofilm development. The topology of the solid interface has been suggested as one of the physical cues influencing bacteria-surface interactions and biofilm development. Using the model organism Bacillus subtilis, we study the transformation of swimming bacteria in liquid culture into robust biofilms in a range of confinement geometries (planar, spherical and toroidal) and interfaces (air/water, silicone/water, and silicone elastomer/water). We find that B. subtilis form submerged biofilms at both solid and liquid interfaces in addition to air-water pellicles. When confined, bacteria grow on curved surfaces of both positive and negative Gaussian curvature. However, the confinement geometry does affect the resulting biofilm roughness and relative coverage. We also find that the biofilm location is governed by oxygen availability as well as by gravitational effects; these compete with each other in some situations. Overall, our results demonstrate that confinement geometry is an effective way to control oxygen availability and subsequently biofilm growth.

Measuring Shape-Dependent Looping Probability of DNA

Recently, several studies have shown that short doubled-stranded DNA (dsDNA) loops more readily than the wormlike chain model predicts. In most of these experiments, the intrinsic bendedness of dsDNA, which in theory can dramatically influence looping dynamics, was either avoided or unaccounted for. To investigate the effect of the shape of dsDNA on looping dynamics, we characterized the shapes of several synthetic dsDNA molecules of equal length but different sequences using gel electrophoresis. We then measured their looping rates using a FRET (Förster resonance energy transfer)-based assay and extracted the looping probability density known as the J factor (jM). We also used, for comparison, several dinucleotide angular parameter sets derived from the observed electrophoretic mobility to compute the jM predicted by the wormlike chain model. Although we found a strong correlation between curvature and jM, the measured jM was higher than most dinucleotide model predictions. This result suggests that it is difficult to reconcile the looping probability with the observed gel mobility within the wormlike chain model and underscores the importance of determining the intrinsic shape of dsDNA for proper theoretical analysis.

Transitions of Double-Stranded DNA Between the A- and B-Forms.

The structure of double-stranded DNA (dsDNA) is sensitive to solvent conditions. In solution, B-DNA is the favored conformation under physiological conditions, while A-DNA is the form found under low water activity. The A-form is induced locally in some protein-DNA complexes, and repeated transitions between the B- and A-forms have been proposed to generate the forces used to drive dsDNA into viral capsids during genome packaging. Here, we report analyses on previous molecular dynamics (MD) simulations on B-DNA, along with new MD simulations on the transition from A-DNA to B-DNA in solution. We introduce the A-B Index (ABI), a new metric along the A-B continuum, to quantify our results. When A-DNA is placed in an equilibrated solution at physiological ionic strength, there is no energy barrier to the transition to the B-form, which begins within about 1 ns. The transition is essentially complete within 5 ns, although occasionally a stretch of a few base pairs will remain A-like for up to ∼10 ns. A comparison of four sequences with a range of predicted A-phobicities shows that more A-phobic sequences make the transition more rapidly than less A-phobic sequences. Simulations on dsDNA with a region of roughly one turn locked in the A-form allow us to characterize the A/B junction, which has an average bend angle of 20-30°. Fluctuations in this angle occur with characteristic times of about 10 ns.

Single-molecule fluorescence studies on DNA looping

Structure and dynamics of DNA impact how the genetic code is processed and maintained. In addition to its biological importance, DNA has been utilized as building blocks of various nanomachines and nanostructures. Thus, understanding the physical properties of DNA is of fundamental importance to basic sciences and engineering applications. DNA can undergo various physical changes. Among them, DNA looping is unique in that it can bring two distal sites together, and thus can be used to mediate interactions over long distances. In this paper, we introduce a FRET-based experimental tool to study DNA looping at the single molecule level. We explain the connection between experimental measurables and a theoretical concept known as the J factor with the intent of raising awareness of subtle theoretical details that should be considered when drawing conclusions. We also explore DNA looping-assisted protein diffusion mechanism called intersegmental transfer using protein induced fluorescence enhancement (PIFE). We present some preliminary results and future outlooks.