Short telomeres induce a DNA damage response, senescence, and apoptosis, thus maintaining telomere length equilibrium is essential for cell viability. Telomerase addition of telomere repeats is tightly regulated in cells. To probe pathways that regulate telomere addition, we developed the ADDIT assay to measure new telomere addition at a single telomere in vivo. Sequence analysis showed telomerase-specific addition of repeats onto a new telomere occurred in just 48 hr. Using the ADDIT assay, we found that ATM is required for addition of new repeats onto telomeres in mouse cells. Evaluation of bulk telomeres, in both human and mouse cells, showed that blocking ATM inhibited telomere elongation. Finally, the activation of ATM through the inhibition of PARP1 resulted in increased telomere elongation, supporting the central role of the ATM pathway in regulating telomere addition. Understanding this role of ATM may yield new areas for possible therapeutic intervention in telomere-mediated disease. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
SecM is an E. coli secretion monitor capable of stalling translation on the prokaryotic ribosome without cofactors. Biochemical and structural studies have demonstrated that the SecM nascent chain interacts with the 50S subunit exit tunnel to inhibit peptide bond formation. However, the timescales and pathways of stalling on an mRNA remain undefined. To provide a dynamic mechanism for stalling, we directly tracked the dynamics of elongation on ribosomes translating the SecM stall sequence (FSTPVWISQAQGIRAGP) using single-molecule fluorescence techniques. Within 1 min, three peptide-ribosome interactions work cooperatively over the last five codons of the SecM sequence, leading to severely impaired elongation rates beginning from the terminal proline and lasting four codons. Our results suggest that stalling is tightly linked to the dynamics of elongation and underscore the roles that the exit tunnel and nascent chain play in controlling fundamental steps in translation. opyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.
The traditional view of macrolide antibiotics as plugs inside the ribosomal nascent peptide exit tunnel (NPET) has lately been challenged in favor of a more complex, heterogeneous mechanism, where drug-peptide interactions determine the fate of a translating ribosome. To investigate these highly dynamic processes, we applied single-molecule tracking of elongating ribosomes during inhibition of elongation by erythromycin of several nascent chains, including ErmCL and H-NS, which were shown to be, respectively, sensitive and resistant to erythromycin. Peptide sequence-specific changes were observed in translation elongation dynamics in the presence of a macrolide-obstructed NPET. Elongation rates were not severely inhibited in general by the presence of the drug; instead, stalls or pauses were observed as abrupt events. The dynamic pathways of nascent-chain-dependent elongation pausing in the presence of macrolides determine the fate of the translating ribosome stalling or readthrough. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.
Inferring antibiotic mechanisms on translation through static structures has been challenging, as biological systems are highly dynamic. Dynamic single-molecule methods are also limited to few simultaneously measurable parameters. We have circumvented these limitations with a multifaceted approach to investigate three structurally distinct aminoglycosides that bind to the aminoacyl-transfer RNA site (A site) in the prokaryotic 30S ribosomal subunit: apramycin, paromomycin, and gentamicin. Using several single-molecule fluorescence measurements combined with structural and biochemical techniques, we observed distinct changes to translational dynamics for each aminoglycoside. While all three drugs effectively inhibit translation elongation, their actions are structurally and mechanistically distinct. Apramycin does not displace A1492 and A1493 at the decoding center, as demonstrated by a solution nuclear magnetic resonance structure, causing only limited miscoding; instead, it primarily blocks translocation. Paromomycin and gentamicin, which displace A1492 and A1493, cause significant miscoding, block intersubunit rotation, and inhibit translocation. Our results show the power of combined dynamics, structural, and biochemical approaches to elucidate the complex mechanisms underlying translation and its inhibition. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.
During translation elongation, the ribosome compositional factors elongation factor G (EF-G; encoded by fusA) and tRNA alternately bind to the ribosome to direct protein synthesis and regulate the conformation of the ribosome. Here, we use single-molecule fluorescence with zero-mode waveguides to directly correlate ribosome conformation and composition during multiple rounds of elongation at high factor concentrations in Escherichia coli. Our results show that EF-G bound to GTP (EF-G-GTP) continuously samples both rotational states of the ribosome, binding with higher affinity to the rotated state. Upon successful accommodation into the rotated ribosome, the EF-G-ribosome complex evolves through several rate-limiting conformational changes and the hydrolysis of GTP, which results in a transition back to the nonrotated state and in turn drives translocation and facilitates release of both EF-G-GDP and E-site tRNA. These experiments highlight the power of tracking single-molecule conformation and composition simultaneously in real time.
In order to coordinate the complex biochemical and structural feat of converting triple-nucleotide codons into their corresponding amino acids, the ribosome must physically manipulate numerous macromolecules including the mRNA, tRNAs, and numerous translation factors. The ribosome choreographs binding, dissociation, physical movements, and structural rearrangements so that they synergistically harness the energy from biochemical processes, including numerous GTP hydrolysis steps and peptide bond formation. Due to the dynamic and complex nature of translation, the large cast of ligands involved, and the large number of possible configurations, tracking the global time evolution or dynamics of the ribosome complex in translation has proven to be challenging for bulk methods. Conventional single-molecule fluorescence experiments on the other hand require low concentrations of fluorescent ligands to reduce background noise. The significantly reduced bimolecular association rates under those conditions limit the number of steps that can be observed within the time window available to a fluorophore. The advent of zero-mode waveguide (ZMW) technology has allowed the study of translation at near-physiological concentrations of labeled ligands, moving single-molecule fluorescence microscopy beyond focused model systems into studying the global dynamics of translation in realistic setups. This chapter reviews the recent works using the ZMW technology to dissect the mechanism of translation initiation and elongation in prokaryotes, including complex processes such as translational stalling and frameshifting. Given the success of the technology, similarly complex biological processes could be studied in near-physiological conditions with the controllability of conventional in vitro experiments. Copyright © 2016 Elsevier Inc. All rights reserved.
N(6)-methylation of adenosine (forming m(6)A) is the most abundant post-transcriptional modification within the coding region of mRNA, but its role during translation remains unknown. Here, we used bulk kinetic and single-molecule methods to probe the effect of m(6)A in mRNA decoding. Although m(6)A base-pairs with uridine during decoding, as shown by X-ray crystallographic analyses of Thermus thermophilus ribosomal complexes, our measurements in an Escherichia coli translation system revealed that m(6)A modification of mRNA acts as a barrier to tRNA accommodation and translation elongation. The interaction between an m(6)A-modified codon and cognate tRNA echoes the interaction between a near-cognate codon and tRNA, because delay in tRNA accommodation depends on the position and context of m(6)A within codons and on the accuracy level of translation. Overall, our results demonstrate that chemical modification of mRNA can change translational dynamics.
Telomerase-mediated telomere elongation provides cell populations with the ability to proliferate indefinitely. Telomerase is capable of recognizing and extending the shortest telomeres in cells; nevertheless, how this mechanism is executed remains unclear. Here, we show that, in the fission yeast Schizosaccharomyces pombe, shortened telomeres are highly transcribed into the evolutionarily conserved long noncoding RNA TERRA A fraction of TERRA produced upon telomere shortening is polyadenylated and largely devoid of telomeric repeats, and furthermore, telomerase physically interacts with this polyadenylated TERRA in vivo We also show that experimentally enhanced transcription of a manipulated telomere promotes its association with telomerase and concomitant elongation. Our data represent the first direct evidence that TERRA stimulates telomerase recruitment and activity at chromosome ends in an organism with human-like telomeres. © 2016 The Authors.