Publications by authors named "Dmitri N Ermolenko"

34 Publications

Making ends meet: New functions of mRNA secondary structure.

Wiley Interdiscip Rev RNA 2021 Mar 29;12(2):e1611. Epub 2020 Jun 29.

Department of Biochemistry & Biophysics and Center for RNA Biology, School of Medicine and Dentistry, University of Rochester, Rochester, New York, USA.

The 5' cap and 3' poly(A) tail of mRNA are known to synergistically regulate mRNA translation and stability. Recent computational and experimental studies revealed that both protein-coding and non-coding RNAs will fold with extensive intramolecular secondary structure, which will result in close distances between the sequence ends. This proximity of the ends is a sequence-independent, universal property of most RNAs. Only low-complexity sequences without guanosines are without secondary structure and exhibit end-to-end distances expected for RNA random coils. The innate proximity of RNA ends might have important biological implications that remain unexplored. In particular, the inherent compactness of mRNA might regulate translation initiation by facilitating the formation of protein complexes that bridge mRNA 5' and 3' ends. Additionally, the proximity of mRNA ends might mediate coupling of 3' deadenylation to 5' end mRNA decay. This article is categorized under: RNA Structure and Dynamics > RNA Structure, Dynamics, and Chemistry RNA Structure and Dynamics > Influence of RNA Structure in Biological Systems Translation > Translation Regulation.
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http://dx.doi.org/10.1002/wrna.1611DOI Listing
March 2021

Extending the Spacing between the Shine-Dalgarno Sequence and P-Site Codon Reduces the Rate of mRNA Translocation.

J Mol Biol 2020 07 13;432(16):4612-4622. Epub 2020 Jun 13.

Department of Biochemistry & Biophysics at School of Medicine and Dentistry and Center for RNA Biology, University of Rochester, Rochester, NY 14642, USA. Electronic address:

By forming base-pairing interactions with the 3' end of 16S rRNA, mRNA Shine-Dalgarno (SD) sequences positioned upstream of open reading frames facilitate translation initiation. During the elongation phase of protein synthesis, intragenic SD-like sequences stimulate ribosome frameshifting and may also slow down ribosome movement along mRNA. Here, we show that the length of the spacer between the SD sequence and P-site codon strongly affects the rate of ribosome translocation. Increasing the spacer length beyond 6 nt destabilizes mRNA-tRNA-ribosome interactions and results in a 5- to 10-fold reduction of the translocation rate. These observations suggest that during translation, the spacer between the SD sequence and P-site codon undergoes structural rearrangements, which slow down mRNA translocation and promote mRNA frameshifting.
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http://dx.doi.org/10.1016/j.jmb.2020.06.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7387212PMC
July 2020

mRNA stem-loops can pause the ribosome by hindering A-site tRNA binding.

Elife 2020 05 19;9. Epub 2020 May 19.

Department of Biochemistry and Biophysics at School of Medicine and Dentistry and Center for RNA Biology, University of Rochester, Rochester, United States.

Although the elongating ribosome is an efficient helicase, certain mRNA stem-loop structures are known to impede ribosome movement along mRNA and stimulate programmed ribosome frameshifting via mechanisms that are not well understood. Using biochemical and single-molecule Förster resonance energy transfer (smFRET) experiments, we studied how frameshift-inducing stem-loops from mRNA and the transcript of Human Immunodeficiency Virus (HIV) perturb translation elongation. We find that upon encountering the ribosome, the stem-loops strongly inhibit A-site tRNA binding and ribosome intersubunit rotation that accompanies translation elongation. Electron cryo-microscopy (cryo-EM) reveals that the HIV stem-loop docks into the A site of the ribosome. Our results suggest that mRNA stem-loops can transiently escape the ribosome helicase by binding to the A site. Thus, the stem-loops can modulate gene expression by sterically hindering tRNA binding and inhibiting translation elongation.
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http://dx.doi.org/10.7554/eLife.55799DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7282821PMC
May 2020

A splice site-sensing conformational switch in U2AF2 is modulated by U2AF1 and its recurrent myelodysplasia-associated mutation.

Nucleic Acids Res 2020 06;48(10):5695-5709

Department of Biochemistry and Biophysics and Center for RNA Biology, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA.

An essential heterodimer of the U2AF1 and U2AF2 pre-mRNA splicing factors nucleates spliceosome assembly at polypyrimidine (Py) signals preceding the major class of 3' splice sites. U2AF1 frequently acquires an S34F-encoding mutation among patients with myelodysplastic syndromes (MDS). The influence of the U2AF1 subunit and its S34F mutation on the U2AF2 conformations remains unknown. Here, we employ single molecule Förster resonance energy transfer (FRET) to determine the influence of wild-type or S34F-substituted U2AF1 on the conformational dynamics of U2AF2 and its splice site RNA complexes. In the absence of RNA, the U2AF1 subunit stabilizes a high FRET value, which by structure-guided mutagenesis corresponds to a closed conformation of the tandem U2AF2 RNA recognition motifs (RRMs). When the U2AF heterodimer is bound to a strong, uridine-rich splice site, U2AF2 switches to a lower FRET value characteristic of an open, side-by-side arrangement of the RRMs. Remarkably, the U2AF heterodimer binds weak, uridine-poor Py tracts as a mixture of closed and open U2AF2 conformations, which are modulated by the S34F mutation. Shifts between open and closed U2AF2 may underlie U2AF1-dependent splicing of degenerate Py tracts and contribute to a subset of S34F-dysregulated splicing events in MDS patients.
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http://dx.doi.org/10.1093/nar/gkaa293DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7261175PMC
June 2020

mRNAs and lncRNAs intrinsically form secondary structures with short end-to-end distances.

Nat Commun 2018 10 18;9(1):4328. Epub 2018 Oct 18.

Department of Biochemistry & Biophysics and Center for RNA Biology, School of Medicine and Dentistry, University of Rochester, Rochester, NY, 14642, USA.

The 5' and 3' termini of RNA play important roles in many cellular processes. Using Förster resonance energy transfer (FRET), we show that mRNAs and lncRNAs have an intrinsic propensity to fold in the absence of proteins into structures in which the 5' end and 3' end are ≤7 nm apart irrespective of mRNA length. Computational estimates suggest that the inherent proximity of the ends is a universal property of most mRNA and lncRNA sequences. Only guanosine-depleted RNA sequences with low sequence complexity are unstructured and exhibit end-to-end distances expected for the random coil conformation of RNA. While the biological implications remain to be explored, short end-to-end distances could facilitate the binding of protein factors that regulate translation initiation by bridging mRNA 5' and 3' ends. Furthermore, our studies provide the basis for measuring, computing and manipulating end-to-end distances and secondary structure in RNA in research and biotechnology.
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http://dx.doi.org/10.1038/s41467-018-06792-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6193969PMC
October 2018

Observation of preQ-II riboswitch dynamics using single-molecule FRET.

RNA Biol 2019 09 30;16(9):1086-1092. Epub 2018 Oct 30.

a Department of Biochemistry & Biophysics and Center for RNA Biology, School of Medicine and Dentistry, University of Rochester , Rochester , NY , USA.

PreQ riboswitches regulate the synthesis of the hypermodified tRNA base queuosine by sensing the pyrrolopyrimidine metabolite preQ. Here, we use single-molecule FRET to interrogate the structural dynamics of apo and preQ-bound states of the preQ-II riboswitch from . We find that the apo-form of the riboswitch spontaneously samples multiple conformations. Magnesium ions and preQ stabilize conformations that sequester the ribosome-binding site of the mRNA within the pseudoknotted structure, thus inhibiting translation initiation. Our results reveal that folding of the preQ-II riboswitch is complex and provide evidence favoring a conformational selection model of effector binding by riboswitches of this class.
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http://dx.doi.org/10.1080/15476286.2018.1536591DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6693549PMC
September 2019

Ensemble and single-molecule FRET studies of protein synthesis.

Methods 2018 03 13;137:37-48. Epub 2017 Dec 13.

Department of Biochemistry and Biophysics & Center for RNA Biology, School of Medicine and Dentistry, University of Rochester, Rochester, NY 14642, United States. Electronic address:

Protein synthesis is a complex, multi-step process that involves large conformational changes of the ribosome and protein factors of translation. Over the last decade, Förster resonance energy transfer (FRET) has become instrumental for studying structural rearrangements of the translational apparatus. Here, we discuss the design of ensemble and single-molecule (sm) FRET assays of translation. We describe a number of experimental strategies that can be used to introduce fluorophores into the ribosome, tRNA, mRNA and protein factors of translation. Alternative approaches to tethering of translation components to the microscope slide in smFRET experiments are also reviewed. Finally, we discuss possible challenges in the interpretation of FRET data and ways to address these challenges.
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http://dx.doi.org/10.1016/j.ymeth.2017.12.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5866757PMC
March 2018

Caught on Camera: Intermediates of Ribosome Recycling.

Structure 2016 12;24(12):2035-2036

Department of Biochemistry and Biophysics & Center for RNA Biology, School of Medicine and Dentistry, University of Rochester, Rochester, NY 14642, USA. Electronic address:

Following termination of protein synthesis, bacterial ribosomes are split into subunits by the joint action of elongation factor G and ribosome recycling factor in the process called ribosome recycling. In this issue of Structure, Fu et al. (2016) describe visualization of transient intermediates of ribosome recycling using time-resolved cryogenic electron microscopy.
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http://dx.doi.org/10.1016/j.str.2016.11.009DOI Listing
December 2016

Structural insights into ribosome translocation.

Wiley Interdiscip Rev RNA 2016 09 27;7(5):620-36. Epub 2016 Apr 27.

Department of Biochemistry and Biophysics & Center for RNA Biology, School of Medicine and Dentistry, University of Rochester, Rochester, NY 14642, USA.

During protein synthesis, tRNA and mRNA are translocated from the A to P to E sites of the ribosome thus enabling the ribosome to translate one codon of mRNA after the other. Ribosome translocation along mRNA is induced by the universally conserved ribosome GTPase, elongation factor G (EF-G) in bacteria and elongation factor 2 (EF-2) in eukaryotes. Recent structural and single-molecule studies revealed that tRNA and mRNA translocation within the ribosome is accompanied by cyclic forward and reverse rotations between the large and small ribosomal subunits parallel to the plane of the intersubunit interface. In addition, during ribosome translocation, the 'head' domain of small ribosomal subunit undergoes forward- and back-swiveling motions relative to the rest of the small ribosomal subunit around the axis that is orthogonal to the axis of intersubunit rotation. tRNA/mRNA translocation is also coupled to the docking of domain IV of EF-G into the A site of the small ribosomal subunit that converts the thermally driven motions of the ribosome and tRNA into the forward translocation of tRNA/mRNA inside the ribosome. Despite recent and enormous progress made in the understanding of the molecular mechanism of ribosome translocation, the sequence of structural rearrangements of the ribosome, EF-G and tRNA during translocation is still not fully established and awaits further investigation. WIREs RNA 2016, 7:620-636. doi: 10.1002/wrna.1354 For further resources related to this article, please visit the WIREs website.
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http://dx.doi.org/10.1002/wrna.1354DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4990484PMC
September 2016

EF-G Activation by Phosphate Analogs.

J Mol Biol 2016 05 8;428(10 Pt B):2248-58. Epub 2016 Apr 8.

Department of Biochemistry and Biophysics & Center for RNA Biology, School of Medicine and Dentistry, University of Rochester, Rochester, NY 14642, USA. Electronic address:

Elongation factor G (EF-G) is a universally conserved translational GTPase that promotes the translocation of tRNA and mRNA through the ribosome. EF-G binds to the ribosome in a GTP-bound form and subsequently catalyzes GTP hydrolysis. The contribution of the ribosome-stimulated GTP hydrolysis by EF-G to tRNA/mRNA translocation remains debated. Here, we show that while EF-G•GDP does not stably bind to the ribosome and induce translocation, EF-G•GDP in complex with phosphate group analogs BeF3(-) and AlF4(-) promotes the translocation of tRNA and mRNA. Furthermore, the rates of mRNA translocation induced by EF-G in the presence of GTP and a non-hydrolyzable analog of GTP, GDP•BeF3(-) are similar. Our results are consistent with the model suggesting that GTP hydrolysis is not directly coupled to mRNA/tRNA translocation. Hence, GTP binding is required to induce the activated, translocation-competent conformation of EF-G while GTP hydrolysis triggers EF-G release from the ribosome.
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http://dx.doi.org/10.1016/j.jmb.2016.03.032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4884529PMC
May 2016

An extended U2AF(65)-RNA-binding domain recognizes the 3' splice site signal.

Nat Commun 2016 Mar 8;7:10950. Epub 2016 Mar 8.

Center for RNA Biology and Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642, USA.

How the essential pre-mRNA splicing factor U2AF(65) recognizes the polypyrimidine (Py) signals of the major class of 3' splice sites in human gene transcripts remains incompletely understood. We determined four structures of an extended U2AF(65)-RNA-binding domain bound to Py-tract oligonucleotides at resolutions between 2.0 and 1.5 Å. These structures together with RNA binding and splicing assays reveal unforeseen roles for U2AF(65) inter-domain residues in recognizing a contiguous, nine-nucleotide Py tract. The U2AF(65) linker residues between the dual RNA recognition motifs (RRMs) recognize the central nucleotide, whereas the N- and C-terminal RRM extensions recognize the 3' terminus and third nucleotide. Single-molecule FRET experiments suggest that conformational selection and induced fit of the U2AF(65) RRMs are complementary mechanisms for Py-tract association. Altogether, these results advance the mechanistic understanding of molecular recognition for a major class of splice site signals.
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http://dx.doi.org/10.1038/ncomms10950DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4786784PMC
March 2016

Initiation factor 2 stabilizes the ribosome in a semirotated conformation.

Proc Natl Acad Sci U S A 2015 Dec 14;112(52):15874-9. Epub 2015 Dec 14.

Department of Biochemistry and Biophysics and Center for RNA Biology, School of Medicine and Dentistry, University of Rochester, Rochester, NY 14642

Intersubunit rotation and movement of the L1 stalk, a mobile domain of the large ribosomal subunit, have been shown to accompany the elongation cycle of translation. The initiation phase of protein synthesis is crucial for translational control of gene expression; however, in contrast to elongation, little is known about the conformational rearrangements of the ribosome during initiation. Bacterial initiation factors (IFs) 1, 2, and 3 mediate the binding of initiator tRNA and mRNA to the small ribosomal subunit to form the initiation complex, which subsequently associates with the large subunit by a poorly understood mechanism. Here, we use single-molecule FRET to monitor intersubunit rotation and the inward/outward movement of the L1 stalk of the large ribosomal subunit during the subunit-joining step of translation initiation. We show that, on subunit association, the ribosome adopts a distinct conformation in which the ribosomal subunits are in a semirotated orientation and the L1 stalk is positioned in a half-closed state. The formation of the semirotated intermediate requires the presence of an aminoacylated initiator, fMet-tRNA(fMet), and IF2 in the GTP-bound state. GTP hydrolysis by IF2 induces opening of the L1 stalk and the transition to the nonrotated conformation of the ribosome. Our results suggest that positioning subunits in a semirotated orientation facilitates subunit association and support a model in which L1 stalk movement is coupled to intersubunit rotation and/or IF2 binding.
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http://dx.doi.org/10.1073/pnas.1520337112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4702994PMC
December 2015

Ribosome subunit joining frozen in time.

Structure 2015 Jun;23(6):977-8

Department of Biochemistry and Biophysics and Center for RNA Biology, School of Medicine and Dentistry, University of Rochester, Rochester, NY 14642, USA. Electronic address:

In this issue of Structure, Chen et al. (2015) report the use of a mixing-spraying method of time-resolved cryogenic electron microscopy, which allowed the progression of ribosomal subunit association to be visualized on the millisecond timescale.
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http://dx.doi.org/10.1016/j.str.2015.05.007DOI Listing
June 2015

Movement of elongation factor G between compact and extended conformations.

J Mol Biol 2015 Jan 15;427(2):454-67. Epub 2014 Nov 15.

Department of Biochemistry and Biophysics and Center for RNA Biology, School of Medicine and Dentistry, University of Rochester, Rochester, NY 14642, USA. Electronic address:

Previous structural studies suggested that ribosomal translocation is accompanied by large interdomain rearrangements of elongation factor G (EF-G). Here, we follow the movement of domain IV of EF-G relative to domain II of EF-G using ensemble and single-molecule Förster resonance energy transfer. Our results indicate that ribosome-free EF-G predominantly adopts a compact conformation that can also, albeit infrequently, transition into a more extended conformation in which domain IV moves away from domain II. By contrast, ribosome-bound EF-G predominantly adopts an extended conformation regardless of whether it is interacting with pretranslocation ribosomes or with posttranslocation ribosomes. Our data suggest that ribosome-bound EF-G may also occasionally sample at least one more compact conformation. GTP hydrolysis catalyzed by EF-G does not affect the relative stability of the observed conformations in ribosome-free and ribosome-bound EF-G. Our data support a model suggesting that, upon binding to a pretranslocation ribosome, EF-G moves from a compact to a more extended conformation. This transition is not coupled to but likely precedes both GTP hydrolysis and mRNA/tRNA translocation.
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http://dx.doi.org/10.1016/j.jmb.2014.11.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4297505PMC
January 2015

Following movement of domain IV of elongation factor G during ribosomal translocation.

Proc Natl Acad Sci U S A 2014 Oct 6;111(42):15060-5. Epub 2014 Oct 6.

Department of Biochemistry and Biophysics and Center for RNA Biology, School of Medicine and Dentistry, University of Rochester, Rochester, NY 14642

Translocation of mRNA and tRNAs through the ribosome is catalyzed by a universally conserved elongation factor (EF-G in prokaryotes and EF-2 in eukaryotes). Previous studies have suggested that ribosome-bound EF-G undergoes significant structural rearrangements. Here, we follow the movement of domain IV of EF-G, which is critical for the catalysis of translocation, relative to protein S12 of the small ribosomal subunit using single-molecule FRET. We show that ribosome-bound EF-G adopts distinct conformations corresponding to the pre- and posttranslocation states of the ribosome. Our results suggest that, upon ribosomal translocation, domain IV of EF-G moves toward the A site of the small ribosomal subunit and facilitates the movement of peptidyl-tRNA from the A to the P site. We found no evidence of direct coupling between the observed movement of domain IV of EF-G and GTP hydrolysis. In addition, our results suggest that the pretranslocation conformation of the EF-G-ribosome complex is significantly less stable than the posttranslocation conformation. Hence, the structural rearrangement of EF-G makes a considerable energetic contribution to promoting tRNA translocation.
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http://dx.doi.org/10.1073/pnas.1410873111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4210333PMC
October 2014

A post-translational regulatory switch on UPF1 controls targeted mRNA degradation.

Genes Dev 2014 Sep;28(17):1900-16

Department of Biochemistry and Biophysics, School of Medicine and Dentistry, University of Rochester, Rochester, New York 14642, USA; Center for RNA Biology, University of Rochester, Rochester, New York 14642, USA;

Nonsense-mediated mRNA decay (NMD) controls the quality of eukaryotic gene expression and also degrades physiologic mRNAs. How NMD targets are identified is incompletely understood. A central NMD factor is the ATP-dependent RNA helicase upframeshift 1 (UPF1). Neither the distance in space between the termination codon and the poly(A) tail nor the binding of steady-state, largely hypophosphorylated UPF1 is a discriminating marker of cellular NMD targets, unlike for premature termination codon (PTC)-containing reporter mRNAs when compared with their PTC-free counterparts. Here, we map phosphorylated UPF1 (p-UPF1)-binding sites using transcriptome-wide footprinting or DNA oligonucleotide-directed mRNA cleavage to report that p-UPF1 provides the first reliable cellular NMD target marker. p-UPF1 is enriched on NMD target 3' untranslated regions (UTRs) along with suppressor with morphogenic effect on genitalia 5 (SMG5) and SMG7 but not SMG1 or SMG6. Immunoprecipitations of UPF1 variants deficient in various aspects of the NMD process in parallel with Förster resonance energy transfer (FRET) experiments reveal that ATPase/helicase-deficient UPF1 manifests high levels of RNA binding and disregulated hyperphosphorylation, whereas wild-type UPF1 releases from nonspecific RNA interactions in an ATP hydrolysis-dependent mechanism until an NMD target is identified. 3' UTR-associated UPF1 undergoes regulated phosphorylation on NMD targets, providing a binding platform for mRNA degradative activities. p-UPF1 binding to NMD target 3' UTRs is stabilized by SMG5 and SMG7. Our results help to explain why steady-state UPF1 binding is not a marker for cellular NMD substrates and how this binding is transformed to induce mRNA decay.
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http://dx.doi.org/10.1101/gad.245506.114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4197951PMC
September 2014

Structure of the ribosome with elongation factor G trapped in the pretranslocation state.

Proc Natl Acad Sci U S A 2013 Dec 9;110(52):20994-9. Epub 2013 Dec 9.

Department of Biochemistry, Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, MA 02454.

During protein synthesis, tRNAs and their associated mRNA codons move sequentially on the ribosome from the A (aminoacyl) site to the P (peptidyl) site to the E (exit) site in a process catalyzed by a universally conserved ribosome-dependent GTPase [elongation factor G (EF-G) in prokaryotes and elongation factor 2 (EF-2) in eukaryotes]. Although the high-resolution structure of EF-G bound to the posttranslocation ribosome has been determined, the pretranslocation conformation of the ribosome bound with EF-G and A-site tRNA has evaded visualization owing to the transient nature of this state. Here we use electron cryomicroscopy to determine the structure of the 70S ribosome with EF-G, which is trapped in the pretranslocation state using antibiotic viomycin. Comparison with the posttranslocation ribosome shows that the small subunit of the pretranslocation ribosome is rotated by ∼12° relative to the large subunit. Domain IV of EF-G is positioned in the cleft between the body and head of the small subunit outwardly of the A site and contacts the A-site tRNA. Our findings suggest a model in which domain IV of EF-G promotes the translocation of tRNA from the A to the P site as the small ribosome subunit spontaneously rotates back from the hybrid, rotated state into the nonrotated posttranslocation state.
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http://dx.doi.org/10.1073/pnas.1311423110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3876248PMC
December 2013

Naked mole-rat has increased translational fidelity compared with the mouse, as well as a unique 28S ribosomal RNA cleavage.

Proc Natl Acad Sci U S A 2013 Oct 30;110(43):17350-5. Epub 2013 Sep 30.

Department of Biology, University of Rochester, Rochester, NY 14627.

The naked mole-rat (Heterocephalus glaber) is a subterranean eusocial rodent with a markedly long lifespan and resistance to tumorigenesis. Multiple data implicate modulation of protein translation in longevity. Here we report that 28S ribosomal RNA (rRNA) of the naked mole-rat is processed into two smaller fragments of unequal size. The two breakpoints are located in the 28S rRNA divergent region 6 and excise a fragment of 263 nt. The excised fragment is unique to the naked mole-rat rRNA and does not show homology to other genomic regions. Because this hidden break site could alter ribosome structure, we investigated whether translation rate and amino acid incorporation fidelity were altered. We report that naked mole-rat fibroblasts have significantly increased translational fidelity despite having comparable translation rates with mouse fibroblasts. Although we cannot directly test whether the unique 28S rRNA structure contributes to the increased fidelity of translation, we speculate that it may change the folding or dynamics of the large ribosomal subunit, altering the rate of GTP hydrolysis and/or interaction of the large subunit with tRNA during accommodation, thus affecting the fidelity of protein synthesis. In summary, our results show that naked mole-rat cells produce fewer aberrant proteins, supporting the hypothesis that the more stable proteome of the naked mole-rat contributes to its longevity.
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http://dx.doi.org/10.1073/pnas.1313473110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3808608PMC
October 2013

Blasticidin S inhibits translation by trapping deformed tRNA on the ribosome.

Proc Natl Acad Sci U S A 2013 Jul 3;110(30):12283-8. Epub 2013 Jul 3.

RNA Therapeutics Institute, Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01605, USA.

The antibiotic blasticidin S (BlaS) is a potent inhibitor of protein synthesis in bacteria and eukaryotes. We have determined a 3.4-Å crystal structure of BlaS bound to a 70S⋅tRNA ribosome complex and performed biochemical and single-molecule FRET experiments to determine the mechanism of action of the antibiotic. We find that BlaS enhances tRNA binding to the P site of the large ribosomal subunit and slows down spontaneous intersubunit rotation in pretranslocation ribosomes. However, the antibiotic has negligible effect on elongation factor G catalyzed translocation of tRNA and mRNA. The crystal structure of the antibiotic-ribosome complex reveals that BlaS impedes protein synthesis through a unique mechanism by bending the 3' terminus of the P-site tRNA toward the A site of the large ribosomal subunit. Biochemical experiments demonstrate that stabilization of the deformed conformation of the P-site tRNA by BlaS strongly inhibits peptidyl-tRNA hydrolysis by release factors and, to a lesser extent, peptide bond formation.
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http://dx.doi.org/10.1073/pnas.1304922110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3725078PMC
July 2013

Antibiotics that bind to the A site of the large ribosomal subunit can induce mRNA translocation.

RNA 2013 Feb 17;19(2):158-66. Epub 2012 Dec 17.

Department of Biochemistry and Biophysics and Center for RNA Biology, School of Medicine and Dentistry, University of Rochester, Rochester, New York 14642, USA.

In the absence of elongation factor EF-G, ribosomes undergo spontaneous, thermally driven fluctuation between the pre-translocation (classical) and intermediate (hybrid) states of translocation. These fluctuations do not result in productive mRNA translocation. Extending previous findings that the antibiotic sparsomycin induces translocation, we identify additional peptidyl transferase inhibitors that trigger productive mRNA translocation. We find that antibiotics that bind the peptidyl transferase A site induce mRNA translocation, whereas those that do not occupy the A site fail to induce translocation. Using single-molecule FRET, we show that translocation-inducing antibiotics do not accelerate intersubunit rotation, but act solely by converting the intrinsic, thermally driven dynamics of the ribosome into translocation. Our results support the idea that the ribosome is a Brownian ratchet machine, whose intrinsic dynamics can be rectified into unidirectional translocation by ligand binding.
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http://dx.doi.org/10.1261/rna.035964.112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3543091PMC
February 2013

Controlled gating and electrical detection of single 50S ribosomal subunits through a solid-state nanopore in a microfluidic chip.

Biosens Bioelectron 2011 Nov 5;29(1):34-9. Epub 2011 Aug 5.

School of Engineering, University of California, Santa Cruz, 1156 High St., Santa Cruz, CA 95064, USA.

We describe analysis and control of 50S ribosomal subunits by a solid-state 45nm diameter nanopore incorporated in a microfluidic chip. When used as a resistive pulse sensor, translocation of single 50S subunits through the nanopore produces current blockades that have a linear dependence on applied voltage. Introduction of individual subunits into the fluidic channel shows a threshold behavior that allows controlled entry of individual 50S ribosomal subunits. The incorporation of nanopores into a larger optofluidic chip system opens possibilities for electrical and optical studies of single ribosomes in well-defined and rapidly variable chemical environments.
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http://dx.doi.org/10.1016/j.bios.2011.07.047DOI Listing
November 2011

mRNA translocation occurs during the second step of ribosomal intersubunit rotation.

Nat Struct Mol Biol 2011 Apr 13;18(4):457-62. Epub 2011 Mar 13.

Center for Molecular Biology of RNA, University of California, Santa Cruz, Santa Cruz, California, USA.

During protein synthesis, mRNA and tRNA undergo coupled translocation through the ribosome in a process that is catalyzed by elongation factor G (EF-G). On the basis of cryo-EM reconstructions, counterclockwise and clockwise rotational movements between the large and small ribosomal subunits have been implicated in a proposed ratcheting mechanism to drive the unidirectional movement of translocation. We used a combination of two fluorescence-based approaches to study the timing of these events, intersubunit fluorescence resonance energy transfer measurements to observe relative rotational movement of the subunits, and a fluorescence quenching assay to monitor translocation of mRNA. Binding of EF-G-GTP first induces rapid counterclockwise intersubunit rotation, followed by a slower, clockwise reversal of the rotational movement. We compared the rates of these movements and found that mRNA translocation occurs during the second, clockwise rotation event, corresponding to the transition from the hybrid state to the classical state.
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http://dx.doi.org/10.1038/nsmb.2011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3079290PMC
April 2011

Following movement of the L1 stalk between three functional states in single ribosomes.

Proc Natl Acad Sci U S A 2009 Feb 3;106(8):2571-6. Epub 2009 Feb 3.

Department of Physics, University of Illinois, 1110 West Green Street, Urbana, IL 61801, USA.

The L1 stalk is a mobile domain of the large ribosomal subunit E site that interacts with the elbow of deacylated tRNA during protein synthesis. Here, by using single-molecule FRET, we follow the real-time dynamics of the L1 stalk and observe its movement relative to the body of the large subunit between at least 3 distinct conformational states: open, half-closed, and fully closed. Pretranslocation ribosomes undergo spontaneous fluctuations between the open and fully closed states. In contrast, posttranslocation ribosomes containing peptidyl-tRNA and deacylated tRNA in the classical P/P and E/E states, respectively, are fixed in the half-closed conformation. In ribosomes with a vacant E site, the L1 stalk is observed either in the fully closed or fully open conformation. Several lines of evidence show that the L1 stalk can move independently of intersubunit rotation. Our findings support a model in which the mobility of the L1 stalk facilitates binding, movement, and release of deacylated tRNA by remodeling the structure of the 50S subunit E site between 3 distinct conformations, corresponding to the E/E vacant, P/E hybrid, and classical states.
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http://dx.doi.org/10.1073/pnas.0813180106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2650305PMC
February 2009

Structural dynamics of the ribosome.

Curr Opin Chem Biol 2008 Dec 9;12(6):674-83. Epub 2008 Oct 9.

Center for Molecular Biology of RNA and Department of Molecular, Cell and Developmental Biology, University of California, Santa Cruz, Santa Cruz, CA 95064, USA.

Protein synthesis is inherently a dynamic process, requiring both small-scale and large-scale movements of tRNA and mRNA. It has long been suspected that these movements might be coupled to conformational changes in the ribosome, and in its RNA moieties in particular. Recently, the nature of ribosome structural dynamics has begun to emerge from a combination of approaches, most notably cryo-EM, X-ray crystallography, and FRET. Ribosome movement occurs both on a grand scale, as in the intersubunit rotational movements that are coupled to tRNA-mRNA translocation, and in intricate localized rearrangements such as those that accompany codon-anticodon recognition and peptide bond formation. In spite of much progress, our understanding of the mechanics of translation is now beset with countless new questions, reflecting the vast molecular architecture of the ribosome itself.
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http://dx.doi.org/10.1016/j.cbpa.2008.08.037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3923522PMC
December 2008

Spontaneous intersubunit rotation in single ribosomes.

Mol Cell 2008 Jun;30(5):578-88

Department of Physics, University of Illinois, 1110 West Green Street, Urbana, IL 61801, USA.

During the elongation cycle, tRNA and mRNA undergo coupled translocation through the ribosome catalyzed by elongation factor G (EF-G). Cryo-EM reconstructions of certain EF-G-containing complexes led to the proposal that the mechanism of translocation involves rotational movement between the two ribosomal subunits. Here, using single-molecule FRET, we observe that pretranslocation ribosomes undergo spontaneous intersubunit rotational movement in the absence of EF-G, fluctuating between two conformations corresponding to the classical and hybrid states of the translocational cycle. In contrast, posttranslocation ribosomes are fixed predominantly in the classical, nonrotated state. Movement of the acceptor stem of deacylated tRNA into the 50S E site and EF-G binding to the ribosome both contribute to stabilization of the rotated, hybrid state. Furthermore, the acylation state of P site tRNA has a dramatic effect on the frequency of intersubunit rotation. Our results provide direct evidence that the intersubunit rotation that underlies ribosomal translocation is thermally driven.
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http://dx.doi.org/10.1016/j.molcel.2008.05.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2491453PMC
June 2008

Elongation factor G stabilizes the hybrid-state conformation of the 70S ribosome.

RNA 2007 Sep 13;13(9):1473-82. Epub 2007 Jul 13.

Center for Molecular Biology of RNA, Department of Molecular, Cell and Developmental Biology, University of California-Santa Cruz 95064, USA.

Following peptide bond formation, transfer RNAs (tRNAs) and messenger RNA (mRNA) are translocated through the ribosome, a process catalyzed by elongation factor EF-G. Here, we have used a combination of chemical footprinting, peptidyl transferase activity assays, and mRNA toeprinting to monitor the effects of EF-G on the positions of tRNA and mRNA relative to the A, P, and E sites of the ribosome in the presence of GTP, GDP, GDPNP, and fusidic acid. Chemical footprinting experiments show that binding of EF-G in the presence of the non-hydrolyzable GTP analog GDPNP or GDP.fusidic acid induces movement of a deacylated tRNA from the classical P/P state to the hybrid P/E state. Furthermore, stabilization of the hybrid P/E state by EF-G compromises P-site codon-anticodon interaction, causing frame-shifting. A deacylated tRNA bound to the P site and a peptidyl-tRNA in the A site are completely translocated to the E and P sites, respectively, in the presence of EF-G with GTP or GDPNP but not with EF-G.GDP. Unexpectedly, translocation with EF-G.GTP leads to dissociation of deacylated tRNA from the E site, while tRNA remains bound in the presence of EF-G.GDPNP, suggesting that dissociation of tRNA from the E site is promoted by GTP hydrolysis and/or EF-G release. Our results show that binding of EF-G in the presence of GDPNP or GDP.fusidic acid stabilizes the ribosomal intermediate hybrid state, but that complete translocation is supported only by EF-G.GTP or EF-G.GDPNP.
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http://dx.doi.org/10.1261/rna.601507DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1950763PMC
September 2007

The antibiotic viomycin traps the ribosome in an intermediate state of translocation.

Nat Struct Mol Biol 2007 Jun 21;14(6):493-7. Epub 2007 May 21.

Department of Molecular, Cell and Developmental Biology and Center for Molecular Biology of RNA, University of California, Santa Cruz, California 95064, USA.

During protein synthesis, transfer RNA and messenger RNA undergo coupled translocation through the ribosome's A, P and E sites, a process catalyzed by elongation factor EF-G. Viomycin blocks translocation on bacterial ribosomes and is believed to bind at the subunit interface. Using fluorescent resonance energy transfer and chemical footprinting, we show that viomycin traps the ribosome in an intermediate state of translocation. Changes in FRET efficiency show that viomycin causes relative movement of the two ribosomal subunits indistinguishable from that induced by binding of EF-G with GDPNP. Chemical probing experiments indicate that viomycin induces formation of a hybrid-state translocation intermediate. Thus, viomycin inhibits translation through a unique mechanism, locking ribosomes in the hybrid state; the EF-G-induced 'ratcheted' state observed by cryo-EM is identical to the hybrid state; and, since translation is viomycin sensitive, the hybrid state may be present in vivo.
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http://dx.doi.org/10.1038/nsmb1243DOI Listing
June 2007

Observation of intersubunit movement of the ribosome in solution using FRET.

J Mol Biol 2007 Jul 20;370(3):530-40. Epub 2007 Apr 20.

Center for Molecular Biology of RNA and Department of Molecular, Cell and Developmental Biology, University of California, Santa Cruz, Santa Cruz, CA 95064, USA.

Protein synthesis is believed to be a dynamic process, involving structural rearrangements of the ribosome. Cryo-EM reconstructions of certain elongation factor G (EF-G)-containing complexes have led to the proposal that translocation of tRNA and mRNA through the ribosome, from the A to P to E sites, is accompanied by a rotational movement between the two ribosomal subunits. Here, we have used Förster resonance energy transfer (FRET) to monitor changes in the relative orientation of the ribosomal subunits in different complexes trapped at intermediate stages of translocation in solution. Binding of EF-G to the ribosome in the presence of the non-hydrolyzable GTP analogue GDPNP or GTP plus fusidic acid causes an increase in the efficiency of energy transfer between fluorophores introduced into proteins S11 in the 30 S subunit and L9 in the 50 S subunit, and a decrease in energy transfer between S6 and L9. Similar anti-correlated changes in energy transfer occur upon binding the GTP-requiring release factor RF3. These changes are consistent with the counter-clockwise rotation of the 30 S subunit relative to the 50 S subunit observed in cryo-EM studies. Reaction of ribosomal complexes containing the peptidyl-tRNA analogues N-Ac-Phe-tRNAPhe, N-Ac-Met-tRNAMet or f-Met-tRNAfMet with puromycin, conditions favoring movement of the resulting deacylated tRNAs into the P/E hybrid state, leads to similar changes in FRET. Conversely, treatment of a ribosomal complex containing deacylated and peptidyl-tRNAs bound in the A/P and P/E states, respectively, with EF-G.GTP causes reversal of the FRET changes. The use of FRET has enabled direct observation of intersubunit movement in solution, provides independent evidence that formation of the hybrid state is coupled to rotation of the 30 S subunit and shows that the intersubunit movement is reversed during the second step of translocation.
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http://dx.doi.org/10.1016/j.jmb.2007.04.042DOI Listing
July 2007

Elimination of the C-cap in ubiquitin - structure, dynamics and thermodynamic consequences.

Biophys Chem 2007 Mar 5;126(1-3):25-35. Epub 2006 Apr 5.

Department of Biochemistry and Molecular Biology, Penn State University College of Medicine, Hershey, PA 17033, USA.

Single amino acid substitutions rarely produce substantial changes in protein structure. Here we show that substitution of the C-cap residue in the alpha-helix of ubiquitin with proline (34P variant) leads to dramatic structural changes. The resulting conformational perturbation extends over the last two turns of the alpha-helix and leads to enhanced flexibility for residues 27-37. Thermodynamic analysis of this ubiquitin variant using differential scanning calorimetry reveals that the thermal unfolding transition remains highly cooperative, exhibiting two-state behavior. Similarities with the wild type in the thermodynamic parameters (heat capacity change upon unfolding and m-value) of unfolding monitored by DSC and chemical denaturation suggests that the 34P variant has comparable buried surface area. The hydrophobic core of 34P variant is not packed as well as that of the wild type protein as manifested by a lower enthalpy of unfolding. The increased mobility of the polypeptide chain of this ubiquitin variant allows the transient opening of the hydrophobic core as evidenced by ANS binding. Taken together, these results suggest exceptional robustness of cooperativity in protein structures.
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http://dx.doi.org/10.1016/j.bpc.2006.03.017DOI Listing
March 2007