Robustly passivated, gold nanoaperture arrays for single-molecule fluorescence microscopy.

The optical confinement generated by metal-based nanoapertures fabricated on a silica substrate has recently enabled single-molecule fluorescence measurements to be performed at physiologically relevant background concentrations of fluorophore-labeled biomolecules. Nonspecific adsorption of fluorophore-labeled biomolecules to the metallic cladding and silica bottoms of nanoapertures, however, remains a critical limitation. To overcome this limitation, we have developed a selective functionalization chemistry whereby the metallic cladding of gold nanoaperture arrays is passivated with methoxy-terminated, thiol-derivatized polyethylene glycol (PEG), and the silica bottoms of those arrays are functionalized with a binary mixture of methoxy- and biotin-terminated, silane-derivatized PEG. This functionalization scheme enables biotinylated target biomolecules to be selectively tethered to the silica nanoaperture bottoms via biotin-streptavidin interactions and reduces the nonspecific adsorption of fluorophore-labeled ligand biomolecules. This, in turn, enables the observation of ligand biomolecules binding to their target biomolecules even under greater than 1 µM background concentrations of ligand biomolecules, thereby rendering previously impracticable biological systems accessible to single-molecule fluorescence investigations.

Dissecting functional cooperation among protein subunits in archaeal RNase P, a catalytic ribonucleoprotein complex.

RNase P catalyzes the Mg(2)(+)-dependent 5'-maturation of precursor tRNAs. Biochemical studies on the bacterial holoenzyme, composed of one catalytic RNase P RNA (RPR) and one RNase P protein (RPP), have helped understand the pleiotropic roles (including substrate/Mg(2+) binding) by which a protein could facilitate RNA catalysis. As a model for uncovering the functional coordination among multiple proteins that aid an RNA catalyst, we use archaeal RNase P, which comprises one catalytic RPR and at least four RPPs. Exploiting our previous finding that these archaeal RPPs function as two binary RPP complexes (POP5•RPP30 and RPP21•RPP29), we prepared recombinant RPP pairs from three archaea and established interchangeability of subunits through homologous/heterologous assemblies. Our finding that archaeal POP5•RPP30 reconstituted with bacterial and organellar RPRs suggests functional overlap of this binary complex with the bacterial RPP and highlights their shared recognition of a phylogenetically-conserved RPR catalytic core, whose minimal attributes we further defined through deletion mutagenesis. Moreover, single-turnover kinetic studies revealed that while POP5•RPP30 is solely responsible for enhancing the RPR's rate of precursor tRNA cleavage (by 60-fold), RPP21•RPP29 contributes to increased substrate affinity (by 16-fold). Collectively, these studies provide new perspectives on the functioning and evolution of an ancient, catalytic ribonucleoprotein.

A highly purified, fluorescently labeled in vitro translation system for single-molecule studies of protein synthesis.

Single-molecule fluorescence resonance energy transfer (smFRET) has emerged as a powerful tool for mechanistic investigations of increasingly complex biochemical systems. Recently, we and others have successfully used smFRET to directly investigate the role of structural dynamics in the function and regulation of the cellular protein synthesis machinery. A significant challenge to these experiments, and to analogous experiments in similarly complex cellular machineries, is the need for specific and efficient fluorescent labeling of the biochemical system at locations that are both mechanistically informative and minimally perturbative to the biological activity. Here, we describe the development of a highly purified, fluorescently labeled in vitro translation system that we have successfully designed for smFRET studies of protein synthesis. The general approaches we outline should be amenable to single-molecule fluorescence studies of other complex biochemical systems.

Solution structure of an archaeal RNase P binary protein complex: formation of the 30-kDa complex between Pyrococcus furiosus RPP21 and RPP29 is accompanied by coupled protein folding and highlights critical features for protein-protein and protein-RNA interactions.

Ribonuclease P (RNase P) is a ribonucleoprotein (RNP) enzyme that catalyzes the Mg(2+)-dependent 5' maturation of precursor tRNAs. In all domains of life, it is a ribozyme: the RNase P RNA (RPR) component has been demonstrated to be responsible for catalysis. However, the number of RNase P protein subunits (RPPs) varies from 1 in bacteria to 9 or 10 in eukarya. The archaeal RPR is associated with at least 4 RPPs, which function in pairs (RPP21-RPP29 and RPP30-POP5). We used solution NMR spectroscopy to determine the three-dimensional structure of the protein-protein complex comprising Pyrococcus furiosus RPP21 and RPP29. We found that the protein-protein interaction is characterized by coupled folding of secondary structural elements that participate in interface formation. In addition to detailing the intermolecular contacts that stabilize this 30-kDa binary complex, the structure identifies surfaces rich in conserved basic residues likely vital for recognition of the RPR and/or precursor tRNA. Furthermore, enzymatic footprinting experiments allowed us to localize the RPP21-RPP29 complex to the specificity domain of the RPR. These findings provide valuable new insights into mechanisms of RNP assembly and serve as important steps towards a three-dimensional model of this ancient RNP enzyme.

Studies on Methanocaldococcus jannaschii RNase P reveal insights into the roles of RNA and protein cofactors in RNase P catalysis.

Ribonuclease P (RNase P), a ribonucleoprotein (RNP) complex required for tRNA maturation, comprises one essential RNA (RPR) and protein subunits (RPPs) numbering one in bacteria, and at least four in archaea and nine in eukarya. While the bacterial RPR is catalytically active in vitro, only select euryarchaeal and eukaryal RPRs are weakly active despite secondary structure similarity and conservation of nucleotide identity in their putative catalytic core. Such a decreased archaeal/eukaryal RPR function might imply that their cognate RPPs provide the functional groups that make up the active site. However, substrate-binding defects might mask the ability of some of these RPRs, such as that from the archaeon Methanocaldococcus jannaschii (Mja), to catalyze precursor tRNA (ptRNA) processing. To test this hypothesis, we constructed a ptRNA-Mja RPR conjugate and found that indeed it self-cleaves efficiently (k(obs), 0.15 min(-1) at pH 5.5 and 55 degrees C). Moreover, one pair of Mja RPPs (POP5-RPP30) enhanced k(obs) for the RPR-catalyzed self-processing by approximately 100-fold while the other pair (RPP21-RPP29) had no effect; both binary RPP complexes significantly reduced the monovalent and divalent ionic requirement. Our results suggest a common RNA-mediated catalytic mechanism in all RNase P and help uncover parallels in RNase P catalysis hidden by plurality in its subunit make-up.

Deciphering RNA structural diversity and systematic phylogeny from microbial metagenomes.

Metagenomics has been employed to systematically sequence, classify, analyze and manipulate the entire genetic material isolated from environmental samples. Finding genes within metagenomic sequences remains a formidable challenge, and noncoding RNA genes other than those encoding rRNA and tRNA are not well annotated in metagenomic projects. In this work, we identify, validate and analyze the genes coding for RNase P RNA (P RNA) from all published metagenomic projects. P RNA is the RNA subunit of a ubiquitous endoribonuclease RNase P that consists of one RNA subunit and one or more protein subunits. The bacterial P RNAs are classified into two types, Type A and Type B, based on the constituents of the structure involved in precursor tRNA binding. Archaeal P RNAs are classified into Type A and Type M, whereas the Type A is ancestral and close to Type A bacterial P RNA. Bacterial and some archaeal P RNAs are catalytically active without protein subunits, capable of cleaving precursor tRNA transcripts to produce their mature 5'-termini. We have found 328 distinctive P RNAs (320 bacterial and 8 archaeal) from all published metagenomics sequences, which led us to expand by 60% the total number of this catalytic RNA from prokaryotes. Surprisingly, all newly identified P RNAs from metagenomics sequences are Type A, i.e. neither Type B bacterial nor Type M archaeal P RNAs are found. We experimentally validate the authenticity of an archaeal P RNA from Sargasso Sea. One of the distinctive features of some new P RNAs is that the P2 stem has kinked nucleotides in its 5' strand. We find that the single nucleotide J2/3 joint region linking the P2 and P3 stem that was used to distinguish a bacterial P RNA from an archaeal one is no longer applicable, i.e. some archaeal P RNAs have only one nucleotide in the J2/3 joint. We also discuss the phylogenetic analysis based on covariance model of P RNA that offers a few advantages over the one based on 16S rRNA.

Functional reconstitution and characterization of Pyrococcus furiosus RNase P.

RNase P, which catalyzes the magnesium-dependent 5'-end maturation of tRNAs in all three domains of life, is composed of one essential RNA and a varying number of protein subunits depending on the source: at least one in bacteria, four in archaea, and nine in eukarya. To address why multiple protein subunits are needed for archaeal/eukaryal RNase P catalysis, in contrast to their bacterial relative, in vitro reconstitution of these holoenzymes is a prerequisite. Using recombinant subunits, we have reconstituted in vitro the RNase P holoenzyme from the thermophilic archaeon Pyrococcus furiosus (Pfu) and furthered our understanding regarding its functional organization and assembly pathway(s). Whereas Pfu RNase P RNA (RPR) alone is capable of multiple turnover, addition of all four RNase P protein (Rpp) subunits to Pfu RPR results in a 25-fold increase in its k(cat) and a 170-fold decrease in K(m). In fact, even in the presence of only one of two specific pairs of Rpps, the RPR displays activity at lower substrate and magnesium concentrations. Moreover, a pared-down, mini-Pfu RNase P was identified with an RPR deletion mutant. Results from our kinetic and footprinting studies on Pfu RNase P, together with insights from recent structures of bacterial RPRs, provide a framework for appreciating the role of multiple Rpps in archaeal RNase P.

Exploring the potential of plant RNase P as a functional genomics tool.

As we trek into the uncharted territories of the genomic era, there is an urgency for the development of approaches for assigning functions to the multitude of uncharacterized genes. Although currently available knock-out methodologies could be used for uncovering the function of newly discovered genes, the mixed outcomes in terms of the success of these approaches in down-regulating gene expression necessitate the development of new functional genomics tools. This chapter describes in detail the experimental method for targeted mRNA degradation inside plant cells by enticing the endogenous and ubiquitous RNase P into recognition of specific mRNAs as non-natural substrates.