Summary Argonaute proteins play a central role in mediating post-transcriptional gene regulation by microRNAs (miRNAs). Argonautes use the nucleotide sequences in miRNAs as guides for identifying target messenger RNAs for repression. Here we used single-molecule FRET to directly visualize how human Argonaute-2 (Ago2) searches for and identifies target sites in RNAs complementary to its miRNA guide. Our results suggest that Ago2 initially scans for target sites with complementarity to nucleotides 2–4 of the miRNA. This initial transient interaction propagates into a stable association when target complementarity extends to nucleotides 2–8. This stepwise recognition process is coupled to lateral diffusion of Ago2 along the target RNA, which promotes target search by enhancing the retention of Ago2 on the RNA. The combined results reveal the mechanisms that Argonaute likely uses to efficiently identify miRNA target sites within the vast and dynamic agglomeration of RNA molecules in the living cell.

Several prokaryotic Argonaute proteins (pAgos) utilize small DNA guides to mediate host defense by targeting invading DNA complementary to the DNA guide. It is unknown how these DNA guides are being generated and loaded onto pAgo. Here, we demonstrate that guide-free Argonaute from Thermus thermophilus (TtAgo) can degrade double-stranded DNA (dsDNA), thereby generating small dsDNA fragments that subsequently are loaded onto TtAgo. Combining single-molecule fluorescence, molecular dynamic simulations, and structural studies, we show that TtAgo loads dsDNA molecules with a preference toward a deoxyguanosine on the passenger strand at the position opposite to the 5' end of the guide strand. This explains why in vivo TtAgo is preferentially loaded with guides with a 5' end deoxycytidine. Our data demonstrate that TtAgo can independently generate and selectively load functional DNA guides.

The fluorescence kinetics in intact photosystem II core particles from the cyanobacterium Thermosynechococcus elongatus have been measured with picosecond resolution at room temperature in open reaction centers. At least two new lifetime components of approximately 2 and 9 ps have been resolved in the kinetics by global analysis in addition to several known longer-lived components (from 42 ps to approximately 2 ns). Kinetic compartment modeling yields a kinetic description in full agreement with the one found recently by femtosecond transient absorption spectroscopy [Holzwarth et al. (2005) submitted to Proc. Natl. Acad. Sci. U.S.A.]. We have for the first time resolved directly the fluorescence spectrum and the kinetics of the equilibrated excited reaction center in intact photosystem II and have found two early radical pairs before the electron is transferred to the quinone Q(A). The apparent lifetime for primary charge separation is 7 ps, that is, by a factor of 8-12 faster than assumed on the basis of earlier analyses. The main component of excited-state decay is 42 ps. The effective primary charge separation rate constant is 170 ns(-)(1), and the secondary electron-transfer rate constant is 112 ns(-)(1). Both electron-transfer steps are reversible. Electron transfer from pheophytin to Q(A) occurs with an apparent overall lifetime of 350 ps. The energy equilibration between the CP43/CP47 antenna and the reaction center occurs with a main apparent lifetime of approximately 1.5 ps and a minor 10 ps lifetime component. Analysis of the overall trapping kinetics based on the theory of energy migration and trapping on lattices shows that the charge separation kinetics in photosystem II is extremely trap-limited and not diffusion-to-the-trap-limited as claimed in several recent papers. These findings support the validity of the assumptions made in deriving the earlier exciton radical pair equilibrium model [Schatz, G. H., Brock, H., and Holzwarth, A. R. (1988) Biophys. J. 54, 397-405].

Proteomic analyses provide essential information on molecular pathways of cellular systems and the state of a living organism. Mass spectrometry is currently the first choice for proteomic analysis. However, the requirement for a large amount of sample renders a small-scale proteomics study challenging. Here, we demonstrate a proof of concept of single-molecule FRET-based protein fingerprinting. We harnessed the AAA+ protease ClpXP to scan peptides. By using donor fluorophore-labeled ClpP, we sequentially read out FRET signals from acceptor-labeled amino acids of peptides. The repurposed ClpXP exhibits unidirectional processing with high processivity and has the potential to detect low-abundance proteins. Our technique is a promising approach for sequencing protein substrates using a small amount of sample.

The fluorescence kinetics of cyanobacterial photosystem II (PSII) core particles with closed reaction centers (RCs) were studied with picosecond resolution. The data are modeled in terms of electron transfer (ET) and associated protein conformational relaxation processes, resolving four different radical pair (RP) states. The target analyses reveal the importance of protein relaxation steps in the ET chain for the functioning of PSII. We also tested previously published data on cyanobacterial PSII with open RCs using models that involved protein relaxation steps as suggested by our data on closed RCs. The rationale for this reanalysis is that at least one short-lived component could not be described in the previous simpler models. This new analysis supports the involvement of a protein relaxation step for open RCs as well. In this model the rate of ET from reduced pheophytin to the primary quinone Q(A) is determined to be 4.1 ns(-1). The rate of initial charge separation is slowed down substantially from approximately 170 ns(-1) in PSII with open RCs to 56 ns(-1) upon reduction of Q(A). However, the free-energy drop of the first RP is not changed substantially between the two RC redox states. The currently assumed mechanistic model, assuming the same early RP intermediates in both states of RC, is inconsistent with the presented energetics of the RPs. Additionally, a comparison between PSII with closed RCs in isolated cores and in intact cells reveals slightly different relaxation kinetics, with a approximately 3.7 ns component present only in isolated cores.