Telomerase is an essential cellular ribonucleoprotein (RNP) that solves the end replication problem and maintains chromosome stability by adding telomeric DNA to the termini of linear chromosomes [1][2][3] . Genetic mutations that abrogate normal assembly of telomerase RNP cause human disease 4 . It is thus of fundamental and medical importance to decipher cellular strategies for telomerase biogenesis, which will require new insights into how specific interactions occur in a precise order along the RNP assembly pathway. Here, we demonstrate a single-molecule approach to dissect the individual assembly steps of telomerase. Direct observation of complex formation in real time revealed two sequential steps of protein-induced RNA folding, establishing a hierarchical RNP assembly mechanism: interaction with the telomerase holoenzyme protein p65 induces structural rearrangement of telomerase RNA, which in turn directs binding of the telomerase reverse transcriptase (TERT) to form the functional ternary complex. This hierarchical assembly process is facilitated by an evolutionarily conserved structural motif within the RNA. These results identify the RNA folding pathway during telomerase biogenesis and define the mechanism of action for an essential telomerase holoenzyme protein.Telomerase RNP functions as a multi-subunit holoenzyme consisting of telomerase RNA, TERT, and additional protein cofactors. Catalytically active telomerase enzyme can be reconstituted from RNA and TERT in rabbit reticulocyte lysate (RRL) wherein general chaperone activities promote RNP assembly 5,6 . However, the endogenous process of telomerase biogenesis appears to require a more specific assembly pathway 7 . Supporting this view, cellular accumulation of telomerase RNP is promoted by a number of specific RNAbinding proteins, including dyskerin in vertebrates, Sm proteins in yeasts, and La-motif proteins in ciliates [8][9][10][11] . In this work, we exploited single-molecule fluorescence resonance energy transfer (FRET) 12-14 to explore the mechanism for telomerase RNP biogenesis, using the ciliate Tetrahymena thermophila as a model system.The Tetrahymena telomerase RNA is a 159 nucleotide transcript (Fig. 1a) that provides a template for telomere synthesis and functions in adapting the polymerase to its specialized taskCorrespondance and requests for materials should be addressed to X.Z., (E-mail: zhuang@chemistry.harvard.edu). Supplementary Information accompanies the paper on www.nature.com/nature. Competing interests statementThe authors declare that they have no competing financial interests. Fig. 1), we strategically placed a FRET donor (Cy3) and acceptor (Cy5) flanking the regions important for interaction with TERT and the holoenzyme protein p65, a La-motif protein that promotes telomerase RNP accumulation in vivo 11 (Fig. 1a). To facilitate real time observation of telomerase RNP assembly, RNA was surface immobilized through an extension of stem II that does not perturb telomerase activity in vitro or in vivo 19 . Standard ...

Telomerase maintains the simple sequence repeats at chromosome ends, protecting cells from genomic rearrangement, proliferative senescence and death. The telomerase reverse transcriptase (TERT) and telomerase RNA (TER) alone can assemble into active enzyme in a heterologous cell extract, but the physiological process of telomerase biogenesis is more complex. The endogenous accumulation of Tetrahymena thermophila TERT and TER requires an additional telomerase holoenzyme protein, p65. Here, we reconstitute this cellular pathway for telomerase ribonucleoprotein biogenesis in vitro. We demonstrate that tandem RNA interaction domains in p65 recognize the sequence of the TER 3′ stem. Notably, the p65-TER complex recruits TERT much more efficiently than does TER alone. Using bacterially expressed p65 and TERT polypeptides, we show that p65 enhances TERT-TER interaction by a mechanism involving a conserved bulge in the protein-bridging TER molecule. These findings reveal a pathway for telomerase holoenzyme biogenesis that preassembles TER for TERT recruitment.Cellular ribonucleoprotein (RNP) biogenesis has gained attention as a dynamic, regulated cascade of events that can be affected in human disease 1-3. RNPs assemble in vivo aided both by assembly chaperones, which contribute transient interactions, and by architectural RNA-binding proteins, which remain integral components of an assembled RNP 4 . Progress in the elucidation of physiological RNP biogenesis mechanisms has been impeded by the limited success in reconstitution of ordered protein and RNA assembly pathways in vitro.TERT and TER can assemble in rabbit reticulocyte lysate (RRL) to form a minimal recombinant telomerase RNP 5 . The physiological process of telomerase enzyme biogenesis, however, seems more complex. In vivo, human TER accumulation and telomere length maintenance are compromised by disease-associated single-residue substitutions in the RNA-binding protein dyskerin 6,7 . In budding yeast, TER accumulation requires RNAbinding proteins from the Sm family 8 . The potentially pleiotropic impact of vertebrate dyskerin or yeast Sm protein alterations complicates the interpretation of their direct roles in telomerase RNP assembly. Additionally, studies of these RNP assembly pathways in vitro are hampered by the inability to reconstitute a dyskerin or Sm RNP efficiently in the absence of other, typically concerted cellular events. In the biological context, it was thus unresolved whether TERT assembles with TER unassisted or only transiently chaperoned, or whether [9][10][11][12] . Genetic depletion of T. thermophila p65 reduces TER accumulation without impact on other small nuclear RNAs, suggesting that in contrast to vertebrate dyskerin or yeast Sm proteins, p65 has a telomerase-specific function in RNP biogenesis 9 . Based on these and additional findings, we proposed a p65-dependent pathway for the physiological biogenesis of T. thermophila telomerase RNP. Here, we test the proposed telomerase RNP assembly pathway by reconstituting the inter...

Monothiol glutaredoxins (mono-Grx) represent a highly evolutionarily conserved class of proteins present in organisms ranging from prokaryotes to humans. Mono-Grxs have been implicated in iron sulfur (FeS) cluster biosynthesis as potential scaffold proteins and in iron homeostasis via an FeS-containing complex with Fra2p (homolog of E. coli BolA) in yeast, and are linked to signal transduction in mammalian systems. However, the function of the mono-Grx in prokaryotes and the nature of an interaction with BolA-like proteins have not been established. Recent genome-wide screens for E. coli genetic interactions reported the synthetic lethality (combination of mutations leading to cell death; mutation of only one of these genes does not) of a grxD mutation when combined with strains defective in FeS cluster biosynthesis (isc operon) functions [Butland, G. et al. (2008) Nature Methods 5, 789–795]. These data connected the only E. coli mono-Grx, GrxD, to a potential role in FeS cluster biosynthesis. We investigated GrxD to uncover the molecular basis of this synthetic lethality and observed that GrxD can form FeS-bound homodimeric and BolA containing heterodimeric complexes. These complexes display substantially different spectroscopic and functional properties, including the ability to act as scaffold proteins for intact FeS cluster transfer to the model [2Fe-2S] acceptor protein E. coli apo-ferredoxin (Fdx), with the homodimer being significantly more efficient. In this work, we functionally dissect the potential cellular roles of GrxD as a component of both homodimeric and heterodimeric complexes, to ultimately uncover if either of these complexes perform functions linked to FeS cluster biosynthesis.

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The lipopolysaccharide biosynthesis pathway is considered an attractive drug target against the rising threat of multi-drug-resistant Gram-negative bacteria. Here, we report two novel small-molecule inhibitors (compounds 1 and 2) of the acyltransferase LpxA, the first enzyme in the lipopolysaccharide biosynthesis pathway. We show genetically that the antibacterial activities of the compounds against efflux-deficient Escherichia coli are mediated by LpxA inhibition. Consistently, the compounds inhibited the LpxA enzymatic reaction in vitro. Intriguingly, using biochemical, biophysical, and structural characterization, we reveal two distinct mechanisms of LpxA inhibition; compound 1 is a substrate-competitive inhibitor targeting apo LpxA, and compound 2 is an uncompetitive inhibitor targeting the LpxA/product complex. Compound 2 exhibited more favorable biological and physicochemical properties than compound 1 and was optimized using structural information to achieve improved antibacterial activity against wild-type E. coli. These results show that LpxA is a promising antibacterial target and imply the advantages of targeting enzyme/product complexes in drug discovery.