We have found that the efficiency of fluorescence resonance energy transfer between Cy3 and Cy5 terminally attached to the 5 ends of a DNA duplex is significantly affected by the relative orientation of the two fluorophores. The cyanine fluorophores are predominantly stacked on the ends of the helix in the manner of an additional base pair, and thus their relative orientation depends on the length of the helix. Observed fluorescence resonance energy transfer (FRET) efficiency depends on the length of the helix, as well as its helical periodicity. By changing the helical geometry from B form double-stranded DNA to A form hybrid RNA/DNA, a marked phase shift occurs in the modulation of FRET efficiency with helix length. Both curves are well explained by the standard geometry of B and A form helices. The observed modulation for both polymers is less than that calculated for a fully rigid attachment of the fluorophores. However, a model involving lateral mobility of the fluorophores on the ends of the helix explains the observed experimental data. This has been further modified to take account of a minor fraction of unstacked fluorophore observed by fluorescent lifetime measurements. Our data unequivocally establish that Fö rster transfer obeys the orientation dependence as expected for a dipole-dipole interaction.cyanine fluorophores ͉ FRET ͉ kappa squared ͉ single-molecule FRET F luorescence resonance energy transfer (FRET) has become widely used to report on distances over the macromolecular scale in biology (1), reviewed in refs. 2-4. The method is highly sensitive, and consequently has been widely exploited in singlemolecule experiments in biological systems. Energy transfer results from dipolar coupling between the transition moments of two fluorophores, and the efficiency of the process (E FRET ) depends on the separation between the donor and acceptor fluorophores, raised to the sixth power. Although such data are frequently interpreted on the assumption of a simple relationship between E FRET and distance, E FRET should also depend on the relative orientation of the transition dipole vectors.The orientation dependence is likely to be most significant where the fluorophores are constrained (5-9). This has been demonstrated experimentally by using a fluorophore that was terminally affixed to duplex DNA by two points of covalent attachment (10), thereby seriously constraining its motion. This situation is not typical of most FRET studies involving nucleic acids. Fluorophores are normally tethered by a single point of attachment, and in theory would be significantly less constrained. But if the fluorophores adopt a rigid manner of attachment to the helix, an orientational dependence could be observed.Cy3 and Cy5 are a commonly used fluorophore pair, especially in single-molecule experiments. Our earlier NMR studies have shown that when these are attached to the 5Ј termini of duplex DNA via a 3-carbon linker to the 5Ј-phosphate they are predominantly stacked onto the ends of the helix in the manner of an additio...

Conformational control of biomolecular activities can reveal functional insights and enable the engineering of novel activities. Here, we show that conformational control through intramolecular crosslinking of a helicase monomer with undetectable unwinding activity converts it into a super-helicase that can unwind thousands of base pairs processively even against a large opposing force. A natural partner that enhances the helicase activity is shown to achieve its stimulating role also by selectively stabilizing the active conformation. Our work provides insight into how nature achieves the regulation of nucleic acid unwinding activity and introduces a monomeric super-helicase without nuclease activities which may be useful for biotechnological applications.

Roughly one third of the proteome is initially destined for the eukaryotic endoplasmic reticulum or the bacterial plasma membrane1. The proper localization of these proteins is mediated by a universally conserved protein targeting machinery, the signal recognition particle (SRP), which recognizes ribosomes carrying signal sequences2–4 and, via interactions with the SRP receptor5,6, delivers them to the protein translocation machinery on the target membrane7. The SRP is an ancient ribonucleoprotein particle containing an essential, elongated SRP RNA whose precise functions have remained elusive. Here, we used single molecule fluorescence microscopy to demonstrate that the SRP-receptor GTPase complex, after initial assembly at the tetraloop end of SRP RNA, travels over 100 Å to the distal end of this RNA where rapid GTP hydrolysis occurs. This movement is negatively regulated by the translating ribosome and, at a later stage, positively regulated by the SecYEG translocon, providing an attractive mechanism to ensure the productive exchange of the targeting and translocation machineries at the ribosome exit site with exquisite spatial and temporal accuracy. Our results show that large RNAs can act as molecular scaffolds that enable the facile exchange of distinct factors and precise timing of molecular events in a complex cellular process; this concept may be extended to similar phenomena in other ribonucleoprotein complexes.

Encapsulation of a biological molecule or a molecular complex in a vesicle provides a means of biofriendly immobilization for single molecule studies and further enables new types of analysis if the vesicles are permeable. We previously reported on using DMPC (dimyristoylphosphatidylcholine) vesicles for realizing porous bioreactors. Here, we describe a different strategy for making porous vesicles using a bacterial pore-forming toxin, α-hemolysin. Using RNA folding as a test case, we demonstrate that protein-based pores can allow exchange of magnesium ions through the vesicle wall while keeping the RNA molecule inside. Flow measurements indicate that the encapsulated RNA molecules rapidly respond to the change in the outside buffer condition. The approach was further tested by coencapsulating a helicase protein and its single-stranded DNA track. The DNA translocation activity of E. coli Rep helicase inside vesicles was fueled by ATP provided outside the vesicle, and a dramatically higher number of translocation cycles could be observed due to the minuscule vesicle volume that facilitates rapid rebinding after dissociation. These pores are known to be stable over a wide range of experimental conditions, especially at various temperatures, which is not possible with the previous method using DMPC vesicles. Moreover, engineered mutants of the utilized toxin can potentially be exploited in the future applications.

In this work, we demonstrate the capability of using lipid vesicles biofunctionalized with protein channels to perform single molecule fluorescence measurements over a biologically relevant temperature range. Lipid vesicles can serve as an ideal nanocontainer for single molecule fluorescence measurements of bio-macromolecules. One serious limitation of the vesicle encapsulation method has been that the lipid membrane is practically impermeable to most ions and small molecules, limiting its application to observing reactions in equilibrium with the initial buffer condition. In order to permeabilize the barrier, Staphylococcal aureus toxin α-hemolysin (aHL) channels have been incorporated into the membrane. These aHL channels have been characterized using single molecule fluorescence resonance energy transfer (smFRET) signals from vesicle encapsulated guanine-rich DNA that folds in G-quadruplex motif as well as from Rep helicase-DNA system. We show that these aHL channels are permeable to monovalent ions and small molecules, such as ATP, over the biologically relevant temperature range (17–37 °C). Ions can efficiently pass through preformed aHL channels in order to initiate DNA folding without any detectable delay. With addition of the cholesterol to the membrane, we also report 35-fold improvement in aHL channel formation efficiency making this approach more practical for wider applications. Finally, the temperature dependent single molecule enzymatic study inside these nanocontainers is demonstrated by measuring Rep helicase repetitive shuttling dynamic along a single stranded DNA at various temperature values. The permeability of the biofriendly nanocontainer over a wide range of temperature would be effectively applied to other surface-based high throughput measurements and sensors beyond the single molecule fluorescence measurements.

The conformational states of Escherichia coli Rep helicase undergoing ATP hydrolysis while bound to a partial-duplex DNA (pdDNA) were studied using single-molecule FRET. Crystallographic studies showed that Rep bound to single-stranded DNA can exist in open and closed conformations that differ in the orientation of the 2B subdomain. FRET measurements between eight Rep mutants donor-labeled at different residues and pdDNA acceptor-labeled at the junction were conducted at each of the four nucleotide states. The positions of donor-labeled residues, based on crystal structure, and FRET measurements between these donor molecules and the acceptor fluorophore at the DNA junction were used to predict the most likely position for the DNA junction using a triangulation algorithm. These predicted junction positions are compared with the crystal structure to determine whether the open or closed conformation is more consistent with the FRET data. Our data revealed that there are two distinct Rep-pdDNA conformations in the ATPγS and ADP states, an unexpected finding. The primary conformation is similar to that observed in nucleotide-free and ADP.Pi states, and the secondary conformation is a novel conformation where the duplex DNA and 2B subdomain moved as a unit by 13 Å relative to the rest of the protein. The primary conformation found in all nucleotide states is consistent with the closed conformation of the crystal structure however; the secondary conformation is a new conformation that has not been observed before. We discuss the possible implications of this newly observed conformation.

Roughly one third of the proteome is initially destined for the eukaryotic endoplasmic reticulum or the bacterial plasma membrane 1 . The proper localization of these proteins is mediated by a universally conserved protein targeting machinery, the signal recognition particle (SRP), which recognizes ribosomes carrying signal sequences 2-4 and, via interactions with the SRP receptor 5,6 , delivers them to the protein translocation machinery on the target membrane 7 . The SRP is an ancient ribonucleoprotein particle containing an essential, elongated SRP RNA whose precise functions have remained elusive. Here, we used single molecule fluorescence microscopy to demonstrate that the SRP-receptor GTPase complex, after initial assembly at the tetraloop end of SRP RNA, travels over 100 Å to the distal end of this RNA where rapid GTP hydrolysis occurs. This movement is negatively regulated by the translating ribosome and, at a later stage, positively regulated by the SecYEG translocon, providing an attractive mechanism to ensure the productive exchange of the targeting and translocation machineries at the ribosome exit site with exquisite spatial and temporal accuracy. Our results show that large RNAs can act as molecular scaffolds that enable the facile exchange of distinct factors and precise timing of molecular events in a complex cellular process; this concept may be extended to similar phenomena in other ribonucleoprotein complexes.Cotranslational protein targeting face fundamental challenges in both spatial and temporal coordination. Spatially, both the SRP 2-4 and SecYEG (or Sec61p) translocon 7 contact the L23 ribosomal protein and the signal sequence, raising puzzling questions about how the translating ribosome is transferred from the targeting to translocation machinery. Temporally, guanosine-5′-triphosphate (GTP) hydrolysis by the SRP-SRP receptor complex, Users may view, print, copy, download and text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: http://www.nature.com/authors/editorial_policies/license.html#terms

The chromatin remodeling complex Isw2 from S. cerevisiae (yIsw2) mobilizes nucleosomes through an ATP-dependent reaction that is coupled to the translocation of the enzyme along intranucleosomal DNA. In this study we demonstrate that yIsw2 is capable of translocating along single-stranded DNA in a reaction that is coupled to ATP hydrolysis. We find that single-stranded DNA translocation by yIsw2 occurs through a series of repeating uniform steps with an overall macroscopic processivity of P ¼ (0.92 5 0.01); this processivity corresponds to an average translocation distance of (24 5 4) nucleotides before dissociation. This processivity corresponds well to the processivity of nucleosome sliding by yIsw2 thus arguing that single-stranded DNA translocation may be fundamental to the double-stranded DNA translocation required for effective nucleosome mobilization by the enzyme. Furthermore, we find that a slow initiation process, following DNA binding, is required to make yIsw2 competent for DNA translocation. We also provide both evidence that this slow initiation process likely corresponds to the second step of a two-step DNA binding mechanism by yIsw2 and a quantitative description of the kinetics of this DNA binding mechanism.