Harry F Noller

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

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Publications (230)2509.8 Total impact

  • Harry F Noller
    RNA 04/2015; 21(4):478-9. DOI:10.1261/rna.049643.115 · 4.62 Impact Factor
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    ABSTRACT: The central dogma of gene expression (DNA to RNA to protein) is universal, but in different domains of life there are fundamental mechanistic differences within this pathway. For example, the canonical molecular signals used to initiate protein synthesis in bacteria and eukaryotes are mutually exclusive. However, the core structures and conformational dynamics of ribosomes that are responsible for the translation steps that take place after initiation are ancient and conserved across the domains of life. We wanted to explore whether an undiscovered RNA-based signal might be able to use these conserved features, bypassing mechanisms specific to each domain of life, and initiate protein synthesis in both bacteria and eukaryotes. Although structured internal ribosome entry site (IRES) RNAs can manipulate ribosomes to initiate translation in eukaryotic cells, an analogous RNA structure-based mechanism has not been observed in bacteria. Here we report our discovery that a eukaryotic viral IRES can initiate translation in live bacteria. We solved the crystal structure of this IRES bound to a bacterial ribosome to 3.8 Å resolution, revealing that despite differences between bacterial and eukaryotic ribosomes this IRES binds directly to both and occupies the space normally used by transfer RNAs. Initiation in both bacteria and eukaryotes depends on the structure of the IRES RNA, but in bacteria this RNA uses a different mechanism that includes a form of ribosome repositioning after initial recruitment. This IRES RNA bridges billions of years of evolutionary divergence and provides an example of an RNA structure-based translation initiation signal capable of operating in two domains of life.
    Nature 02/2015; 519(7541). DOI:10.1038/nature14219 · 42.35 Impact Factor
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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 09/2014; 345(6201):1188-91. DOI:10.1126/science.1255030 · 31.48 Impact Factor
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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 09/2014; 111(37). DOI:10.1073/pnas.1413731111 · 9.81 Impact Factor
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    ABSTRACT: eLife digest Producing a protein first requires its gene to be transcribed into a long molecule called a messenger RNA (mRNA). A complex molecular machine called the ribosome then translates the mRNA code by reading it three letters at a time. Each triplet of letters—known as a codon—tells the ribosome which amino acid to add next into the protein. After adding an amino acid, the ribosome moves along the mRNA molecule to read the next codon and add another amino acid into the protein chain. While researchers understand how protein chains are formed, how the ribosome shifts along the mRNA strand—a process called translocation—is still unclear. It is known that this process involves many force-generating movements and changes to the shape of the ribosome. However, it is only recently that researchers have been able to measure these forces. Using optical tweezers—an instrument that uses a highly focused laser beam to hold and manipulate microscopic objects—Liu, Kaplan et al. followed individual ribosomes as they translated an mRNA and measured the effect that applying an opposing force has on the rate of translation. The results shed new light on the mechanism of translocation. First, Liu, Kaplan et al. found that ribosomes jump directly from one triplet to the next in the mRNA sequence, rather than moving there in a series of smaller steps. Next, the results indicate that translocation occurs spontaneously, driven by thermal energy, while chemical reactions prevent the reverse movement, in a mechanism known as a ‘Brownian Ratchet’. Measurements of the maximum force generated by the ribosome also give insights into how translation is regulated. Strands of mRNA can fold into certain structures that slow down translation, because the mRNA must first be unfolded before the ribosome can translate it. Liu, Kaplan et al. found that the maximum force generated by a ribosome is only just enough to unwind these mRNA structures, making the translation rate highly sensitive to the existence of such structures, and the structures themselves of high importance for regulating transcription. Given its importance as the ultimate decoder of the genetic information, understanding the ribosome's function and regulation has broad implications. The work of Liu, Kaplan et al. opens the way for a full characterization of the role of mechanical forces in the translation process. DOI: http://dx.doi.org/10.7554/eLife.03406.002
    eLife Sciences 08/2014; 3:e03406. DOI:10.7554/eLife.03406 · 8.52 Impact Factor
  • Experimental Biology Meeting; 04/2014
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    Harry F Noller
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    ABSTRACT: Not long after my arrival at UCSC as an assistant professor, I came across Carl Woese's paper “Molecular Mechanics of Translation: A Reciprocating Ratchet Mechanism.”1 In the days before the crystal structure of tRNA was known, Fuller and Hodgson2 had proposed two alternative conformations for its anticodon loop; one was stacked on the 3′ side (as later found in the crystal structure) and the other on the 5′ side. In an ingenious and elegant model, Woese proposed that the conformation of the loop flips between Fuller and Hodgson's 5′- and 3′-stacked forms during protein synthesis, changing the local direction of the mRNA such that the identities of the tRNA binding sites alternated between binding aminoacyl-tRNA and peptidyl-tRNA. The model predicted that there are no A and P sites, only two binding sites whose identities changed following translation of each codon, and that there would be no translocation of tRNAs in the usual sense—only binding and release. I met Carl in person the following year when he presented a seminar on his ratchet model in Santa Cruz. He was chatting in my colleague Ralph Hinegardner's office in what Carl termed a “Little Jack Horner appointment” (the visitor sits and listens to his host describing “What a good boy am I”). He was of compact stature, and bore a striking resemblance to Oskar Werner in Truffaut's film “Jules and Jim.” He projected the impression of a New-Age guru—a shiny black amulet suspended over the front of his black turtleneck sweater and a crown of prematurely white hair. Ralph asked me to explain to Carl what we were doing with ribosomes. I quickly summarized our early experiments that were pointing to a functional role for 16S rRNA. Carl regarded me silently, with a penetrating stare. He then turned to Ralph and said, in an ominous low voice, “I'm going to have some more tanks made as soon as I get back.” Carl's beautiful model was, unfortunately, wrong—it was simpler and more elegant than the complex mechanism that Nature actually uses. Unyielding, Carl railed against the A-site-P-site model at every opportunity,3,4 and although we ended up enjoying a long, intense, and fruitful collaboration, and became close, life-long friends, I finally gave up trying to describe to him our biochemical and crystallographic results on the A, P, and E sites.
    RNA biology 03/2014; 11(3). DOI:10.4161/rna.27970 · 5.38 Impact Factor
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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; 24(1). DOI:10.1016/j.sbi.2014.01.002 · 8.75 Impact Factor
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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; 110(52). DOI:10.1073/pnas.1320387110 · 9.81 Impact Factor
  • Harry F Noller
    Journal of Molecular Biology 07/2013; 366(20). DOI:10.1016/j.jmb.2013.07.003 · 4.33 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. DOI:10.1126/science.1236086 · 31.48 Impact Factor
  • Harry F Noller
    Journal of Biological Chemistry 06/2013; DOI:10.1074/jbc.X113.497511 · 4.57 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; 21. DOI:10.1016/j.str.2013.04.028 · 6.79 Impact Factor
  • Harry Noller
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    ABSTRACT: Discoverer of life's third domain, the Archaea.
    Nature 01/2013; 493(7434):610. DOI:10.1038/493610a · 42.35 Impact Factor
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    Biophysical Journal 01/2013; 104(2):258a. DOI:10.1016/j.bpj.2012.11.1449 · 3.97 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; DOI:10.1261/rna.035964.112 · 4.62 Impact Factor
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    ABSTRACT: Termination is a crucial step in translation, most notably because premature termination can lead to toxic truncated polypeptides. Most interesting is the fact that stop codons are read by a completely different mechanism from that of sense codons. In recent years, rapid progress has been made in the structural biology of complexes of bacterial ribosomes bound to translation termination factors, much of which has been discussed in earlier reviews [1-5]. Here, we present a brief overview of the structures of bacterial translation termination complexes. The first part summarizes what has been learned from crystal structures of complexes containing the class I release factors RF1 and RF2. In the second part, we discuss the results and implications of two recent X-ray structures of complexes of ribosomes bound to the translational GTPase RF3. These structures have provided many insights and a number of surprises. While structures alone do not tell us how these complicated molecular assemblies work, is it nevertheless clear that it will not be possible to understand their mechanisms without detailed structural information.
    Current Opinion in Structural Biology 12/2012; 22(6):733–742. DOI:10.1016/j.sbi.2012.08.004 · 8.75 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; 109(50). DOI:10.1073/pnas.1218999109 · 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. DOI:10.1073/pnas.1208950109 · 9.81 Impact Factor

Publication Stats

23k Citations
2,509.80 Total Impact Points

Institutions

  • 1974–2015
    • University of California, Santa Cruz
      • • Department of Molecular Cell & Developmental Biology
      • • Center for the Molecular Biology of RNA
      Santa Cruz, California, United States
  • 2014
    • Max Planck Institute of Molecular Cell Biology and Genetics
      Dresden, Saxony, Germany
  • 2012
    • University of Rochester
      • School of Medicine and Dentistry
      Rochester, NY, United States
  • 1982–2008
    • University of California, Berkeley
      • Department of Chemistry
      Berkeley, MO, United States
    • Harvard University
      Cambridge, Massachusetts, 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
      • Department of Biochemistry, Biophysics and Molecular Biology
      Ames, Iowa, United States
  • 1986–1997
    • University of California Observatories
      Santa Cruz, California, United States
  • 1995
    • University of Vienna
      Wien, Vienna, Austria
  • 1994
    • Stanford University
      Palo Alto, California, United States