H F Noller

University of California, Santa Cruz, Santa Cruz, CA, United States

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Publications (218)2252.49 Total impact

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    ABSTRACT: Coupled translocation of messenger RNA and transfer RNA (tRNA) through the ribosome, a process catalyzed by elongation factor EF-G, is a crucial step in protein synthesis. The crystal structure of a bacterial translocation complex describes the binding states of two tRNAs trapped in mid-translocation. The deacylated P-site tRNA has moved into a partly translocated pe/E chimeric hybrid state. The anticodon stem-loop of the A-site tRNA is captured in transition toward the 30S P site, while its 3' acceptor end contacts both the A and P loops of the 50S subunit, forming an ap/ap chimeric hybrid state. The structure shows how features of ribosomal RNA rearrange to hand off the A-site tRNA to the P site, revealing an active role for ribosomal RNA in the translocation process.
    Science (New York, N.Y.). 09/2014; 345(6201):1188-91.
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    ABSTRACT: During ribosomal translocation, a process central to the elongation phase of protein synthesis, movement of mRNA and tRNAs requires large-scale rotation of the head domain of the small (30S) subunit of the ribosome. It has generally been accepted that the head rotates by pivoting around the neck helix (h28) of 16S rRNA, its sole covalent connection to the body domain. Surprisingly, we observe that the calculated axis of rotation does not coincide with the neck. Instead, comparative structure analysis across 55 ribosome structures shows that 30S head movement results from flexing at two hinge points lying within conserved elements of 16S rRNA. Hinge 1, although located within the neck, moves by straightening of the kinked helix h28 at the point of contact with the mRNA. Hinge 2 lies within a three-way helix junction that extends to the body through a second, noncovalent connection; its movement results from flexing between helices h34 and h35 in a plane orthogonal to the movement of hinge 1. Concerted movement at these two hinges accounts for the observed magnitudes of head rotation. Our findings also explain the mode of action of spectinomycin, an antibiotic that blocks translocation by binding to hinge 2.
    Proceedings of the National Academy of Sciences of the United States of America. 09/2014;
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    ABSTRACT: A system for naming ribosomal proteins is described that the authors intend to use in the future. They urge others to adopt it. The objective is to eliminate the confusion caused by the assignment of identical names to ribosomal proteins from different species that are unrelated in structure and function. In the system proposed here, homologous ribosomal proteins are assigned the same name, regardless of species. It is designed so that new names are similar enough to old names to be easily recognized, but are written in a format that unambiguously identifies them as 'new system' names.
    Current Opinion in Structural Biology 02/2014; · 8.74 Impact Factor
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    ABSTRACT: A detailed understanding of tRNA/mRNA translocation requires measurement of the forces generated by the ribosome during this movement. Such measurements have so far remained elusive and, thus, little is known about the relation between force and translocation and how this reflects on its mechanism and regulation. Here, we address these questions using optical tweezers to follow translation by individual ribosomes along single mRNA molecules, against an applied force. We find that translocation rates depend exponentially on the force, with a characteristic distance close to the one-codon step, ruling out the existence of sub-steps and showing that the ribosome likely functions as a Brownian ratchet. We show that the ribosome generates ∼13 pN of force, barely sufficient to unwind the most stable structures in mRNAs, thus providing a basis for their regulatory role. Our assay opens the way to characterizing the ribosome's full mechano-chemical cycle.DOI: http://dx.doi.org/10.7554/eLife.03406.001.
    eLife. 01/2014; 3:e03406.
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    ABSTRACT: During protein synthesis, coupled translocation of messenger RNAs (mRNA) and transfer RNAs (tRNA) through the ribosome takes place following formation of each peptide bond. The reaction is facilitated by large-scale conformational changes within the ribosomal complex and catalyzed by elongtion factor G (EF-G). Previous structural analysis of the interaction of EF-G with the ribosome used either model complexes containing no tRNA or only a single tRNA, or complexes where EF-G was directly bound to ribosomes in the posttranslocational state. Here, we present a multiparticle cryo-EM reconstruction of a translocation intermediate containing two tRNAs trapped in transit, bound in chimeric intrasubunit ap/P and pe/E hybrid states. The downstream ap/P-tRNA is contacted by domain IV of EF-G and P-site elements within the 30S subunit body, whereas the upstream pe/E-tRNA maintains tight interactions with P-site elements of the swiveled 30S head. Remarkably, a tight compaction of the tRNA pair can be seen in this state. The translocational intermediate presented here represents a previously missing link in understanding the mechanism of translocation, revealing that the ribosome uses two distinct molecular ratchets, involving both intra- and intersubunit rotational movements, to drive the synchronous movement of tRNAs and mRNA.
    Proceedings of the National Academy of Sciences 12/2013; · 9.81 Impact Factor
  • Harry F Noller
    Journal of Molecular Biology 07/2013; · 3.91 Impact Factor
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    ABSTRACT: Translocation of messenger and transfer RNA (mRNA and tRNA) through the ribosome is a crucial step in protein synthesis, whose mechanism is not yet understood. The crystal structures of three Thermus ribosome-tRNA-mRNA-EF-G complexes trapped with β,γ-imidoguanosine 5'-triphosphate (GDPNP) or fusidic acid reveal conformational changes occurring during intermediate states of translocation, including large-scale rotation of the 30S subunit head and body. In all complexes, the tRNA acceptor ends occupy the 50S subunit E site, while their anticodon stem loops move with the head of the 30S subunit to positions between the P and E sites, forming chimeric intermediate states. Two universally conserved bases of 16S ribosomal RNA that intercalate between bases of the mRNA may act as "pawls" of a translocational ratchet. These findings provide new insights into the molecular mechanism of ribosomal translocation.
    Science 06/2013; 340(6140):1236086. · 31.20 Impact Factor
  • Harry F Noller
    Journal of Biological Chemistry 06/2013; · 4.65 Impact Factor
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    ABSTRACT: Bacterial translation termination is mediated by release factors RF1 and RF2, which recognize stop codons and catalyze hydrolysis of the peptidyl-tRNA ester bond. The catalytic mechanism has been debated. We proposed that the backbone amide NH group, rather than the side chain, of the glutamine of the universally conserved GGQ motif participates in catalysis by H-bonding to the tetrahedral transition-state intermediate and by product stabilization. This was supported by complete loss of RF1 catalytic activity when glutamine is replaced by proline, the only residue that lacks a backbone NH group. Here, we present the 3.4 Å crystal structure of the ribosome complex containing the RF2 Q253P mutant and find that its fold, including the GGP sequence, is virtually identical to that of wild-type RF2. This rules out proline-induced misfolding and further supports the proposal that catalytic activity requires interaction of the Gln-253 backbone amide with the 3' end of peptidyl-tRNA.
    Structure 06/2013; · 5.99 Impact Factor
  • Harry Noller
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    ABSTRACT: Discoverer of life's third domain, the Archaea.
    Nature 01/2013; 493(7434):610. · 38.60 Impact Factor
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    ABSTRACT: 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.
    RNA 12/2012; · 5.09 Impact Factor
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    ABSTRACT: Highlights ► Stop codons are recognized directly by the class I release factors RF1 and RF2. ► Peptidyl-tRNA hydrolysis is catalyzed by the class I release factors themselves. ► The backbone amide nitrogen of the Q in the GGQ motif is involved in catalysis. ► The class II release factor RF3, a GTPase related to EF-G, induces large-scale rotations of the 30S body and head. ► The structure of the RF3·GDPNP·ribosome complex has potential implications for the mechanism of action of EF-G.
    Current Opinion in Structural Biology 12/2012; 22(6):733–742. · 8.74 Impact Factor
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    Zhuojun Guo, Harry F Noller
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    ABSTRACT: Elongation factor-G-catalyzed translocation of mRNA and tRNAs during protein synthesis involves large-scale conformational changes in the ribosome. Formation of hybrid-state intermediates is coupled to counterclockwise (forward) rotation of the body of the 30S subunit. Recent structural studies implicate intrasubunit rotation of the 30S head in translocation. Here, we observe rotation of the head during translocation in real time using ensemble stopped-flow FRET with ribosomes containing fluorescent probes attached to specific positions in the head and body of the 30S subunit. Our results allow ordering of the rates of movement of the 30S subunit body and head during translocation: body forward > head forward > head reverse ≥ body reverse. The rate of quenching of pyrene-labeled mRNA is consistent with coupling of mRNA translocation to head rotation.
    Proceedings of the National Academy of Sciences 11/2012; · 9.81 Impact Factor
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    ABSTRACT: The sequence and secondary structure of the 5'-end of mRNAs regulate translation by controlling ribosome initiation on the mRNA. Ribosomal protein S1 is crucial for ribosome initiation on many natural mRNAs, particularly for those with structured 5'-ends, or with no or weak Shine-Dalgarno sequences. Besides a critical role in translation, S1 has been implicated in several other cellular processes, such as transcription recycling, and the rescuing of stalled ribosomes by tmRNA. The mechanisms of S1 functions are still elusive but have been widely considered to be linked to the affinity of S1 for single-stranded RNA and its corresponding destabilization of mRNA secondary structures. Here, using optical tweezers techniques, we demonstrate that S1 promotes RNA unwinding by binding to the single-stranded RNA formed transiently during the thermal breathing of the RNA base pairs and that S1 dissociation results in RNA rezipping. We measured the dependence of the RNA unwinding and rezipping rates on S1 concentration, and the force applied to the ends of the RNA. We found that each S1 binds 10 nucleotides of RNA in a multistep fashion implying that S1 can facilitate ribosome initiation on structured mRNA by first binding to the single strand next to an RNA duplex structure ("stand-by site") before subsequent binding leads to RNA unwinding. Unwinding by multiple small substeps is much less rate limited by thermal breathing than unwinding in a single step. Thus, a multistep scheme greatly expedites S1 unwinding of an RNA structure compared to a single-step mode.
    Proceedings of the National Academy of Sciences 08/2012; 109(36):14458-63. · 9.81 Impact Factor
  • Biophysical Journal 01/2012; 102(3):68-. · 3.67 Impact Factor
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    ABSTRACT: The class II release factor RF3 is a GTPase related to elongation factor EF-G, which catalyzes release of class I release factors RF1 and RF2 from the ribosome after termination of protein synthesis. The 3.3 Å crystal structure of the RF3·GDPNP·ribosome complex provides a high-resolution description of interactions and structural rearrangements that occur when binding of this translational GTPase induces large-scale rotational movements in the ribosome. RF3 induces a 7° rotation of the body and 14° rotation of the head of the 30S ribosomal subunit, and itself undergoes inter- and intradomain conformational rearrangements. We suggest that ordering of critical elements of switch loop I and the P loop, which help to form the GTPase catalytic site, are caused by interactions between the G domain of RF3 and the sarcin-ricin loop of 23S rRNA. The rotational movements in the ribosome induced by RF3, and its distinctly different binding orientation to the sarcin-ricin loop of 23S rRNA, raise interesting implications for the mechanism of action of EF-G in translocation.
    RNA 12/2011; 18(2):230-40. · 5.09 Impact Factor
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    Michael D Pearson, Harry F Noller
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    ABSTRACT: The Planococcaceae are extreme survivors, having been cultured from environments such as deep sea sediments, marine solar salterns, glaciers, permafrost, Antarctic deserts, and sea ice brine. The family contains both sporulating and nonsporulating genera. Here we present the unclosed, draft genome sequence of Planococcus donghaensis strain MPA1U2, a nonsporulating psychrotrophic bacterium isolated from surface coastal water of the Pacific Ocean.
    Journal of bacteriology 11/2011; 193(21):6106. · 3.94 Impact Factor
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    ABSTRACT: 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.
    Biosensors & bioelectronics 08/2011; 29(1):34-9. · 5.43 Impact Factor
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    ABSTRACT: The ribosome translates the genetic information encoded in messenger RNA into protein. Folded structures in the coding region of an mRNA represent a kinetic barrier that lowers the peptide elongation rate, as the ribosome must disrupt structures it encounters in the mRNA at its entry site to allow translocation to the next codon. Such structures are exploited by the cell to create diverse strategies for translation regulation, such as programmed frameshifting, the modulation of protein expression levels, ribosome localization and co-translational protein folding. Although strand separation activity is inherent to the ribosome, requiring no exogenous helicases, its mechanism is still unknown. Here, using a single-molecule optical tweezers assay on mRNA hairpins, we find that the translation rate of identical codons at the decoding centre is greatly influenced by the GC content of folded structures at the mRNA entry site. Furthermore, force applied to the ends of the hairpin to favour its unfolding significantly speeds translation. Quantitative analysis of the force dependence of its helicase activity reveals that the ribosome, unlike previously studied helicases, uses two distinct active mechanisms to unwind mRNA structure: it destabilizes the helical junction at the mRNA entry site by biasing its thermal fluctuations towards the open state, increasing the probability of the ribosome translocating unhindered; and it mechanically pulls apart the mRNA single strands of the closed junction during the conformational changes that accompany ribosome translocation. The second of these mechanisms ensures a minimal basal rate of translation in the cell; specialized, mechanically stable structures are required to stall the ribosome temporarily. Our results establish a quantitative mechanical basis for understanding the mechanism of regulation of the elongation rate of translation by structured mRNAs.
    Nature 07/2011; 475(7354):118-21. · 38.60 Impact Factor

Publication Stats

18k Citations
2,252.49 Total Impact Points

Institutions

  • 1974–2013
    • University of California, Santa Cruz
      • • Center for the Molecular Biology of RNA
      • • Department of Molecular Cell & Developmental Biology
      Santa Cruz, CA, United States
  • 2012
    • University of Rochester
      • School of Medicine and Dentistry
      Rochester, NY, United States
  • 2008–2012
    • University of California, Berkeley
      • Department of Chemistry
      Berkeley, CA, United States
  • 2002
    • Molecular and Cellular Biology Program
      Seattle, Washington, United States
  • 2000
    • University of California, San Diego
      • Department of Chemistry and Biochemistry
      San Diego, CA, United States
    • Iowa State University
      Ames, Iowa, United States
  • 1999
    • University of Illinois at Chicago
      • Center for Pharmaceutical Biotechnology
      Chicago, IL, United States
  • 1997
    • University of Vienna
      Wien, Vienna, Austria
  • 1994
    • Stanford University
      Palo Alto, California, United States
  • 1991
    • King's College London
      • Department of Biochemistry
      London, ENG, United Kingdom
  • 1989
    • Odense University Hospital
      • Molecular biology laboratory
      Odense, South Denmark, Denmark
  • 1986
    • University of Illinois, Urbana-Champaign
      • Department of Microbiology
      Urbana, IL, United States
  • 1981
    • CSU Mentor
      Long Beach, California, United States
  • 1979
    • University of California, Davis
      Davis, California, United States