Harry Noller

RNA modeling using Gibbs sampling and stochastic context free grammars.

Published in International Conference on Intelligent Systems for Molecular Biology

A new method of discovering the common secondary structure of a family of homologous RNA sequences using Gibbs sampling and stochastic context-free grammars is proposed. Given an unaligned set of sequences, a Gibbs sampling step simultaneously estimates the secondary structure of each sequence and a set of statistical parameters describing the comm...

Identification of bases in 16S rRNA essential for tRNA binding at the 30S ribosomal P site.

Published in Science

Previous studies suggest that the mechanism of action of the ribosome in translation involves crucial transfer RNA (tRNA)-ribosomal RNA (rRNA) interactions. Here, a selection scheme was developed to identify bases in 16S rRNA that are essential for tRNA binding to the P site of the small (30S) ribosomal subunit. Modification of the N-1 and N-2 posi...

Molecular mechanics of 30S subunit head rotation.

Published in Proceedings of the National Academy of Sciences

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...

Allele-specific structure probing of plasmid-derived 16S ribosomal RNA from Escherichia coli.

Published in Gene

Biochemical analysis of Escherichia coli ribosomes containing mutant 16S or 23S (r)ribosomal RNAs, produced via cloned rDNA genes on multicopy plasmids, has been hindered by the background of wild-type (wt) ribosomes containing host-derived rRNA. Here, we describe a method for the construction of unique priming sites in 16S rRNA that allow allele-s...

Direct measurement of the mechanical work during translocation by the ribosome.

Published in eLife

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 us...

Accurate translocation of mRNA by the ribosome requires a peptidyl group or its analog on the tRNA moving into the 30S P...

Published in Molecular Cell

The ribosome must accurately translocate mRNA to maintain the reading frame. Here, we monitor the position of mRNA within the ribosome before and after EF-G-catalyzed translocation near the initiation site. When a deacylated tRNA that is translocated to the 30S P site recognizes other nearby codons, movement of tRNA and mRNA often becomes uncoupled...

Methylation interference experiments identify bases that are essential for distinct catalytic functions of a group I rib...

Published in The EMBO Journal

Methylation interference experiments reveal bases involved in three different catalytic functions of the T4-phage derived sunY self-splicing intron. RNA molecules methylated at the N-7 position of the guanine at the cofactor binding site are inactive in cofactor-dependent splicing and 3 splice-site hydrolysis. In contrast, 5 splice-site hydrolysi...

mRNA helicase activity of the ribosome.

Published in Cell

Most mRNAs contain secondary structure, yet their codons must be in single-stranded form to be translated. Until now, no helicase activity has been identified which could account for the ability of ribosomes to translate through downstream mRNA secondary structure. Using an oligonucleotide displacement assay, together with a stepwise in vitro trans...

Biochemical characterization of the ribosomal decoding site.

Published in Biochimie

Prior to the emergence of crystal structures of the ribosome, different ribosomal functions were identified with specific regions of ribosomal RNA by biochemical and genetic approaches. In particular, three universally conserved bases of 16S rRNA, G530, A1492 and A1493, were implicated in the interaction of the incoming aminoacyl-tRNA with the 30S ...

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

Published in Nature Structural & Molecular Biology

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, w...

Orientation of ribosome recycling factor in the ribosome from directed hydroxyl radical probing.

Published in Cell

Ribosome recycling factor (RRF) disassembles posttermination complexes in conjunction with elongation factor EF-G, liberating ribosomes for further rounds of translation. The striking resemblance of its L-shaped structure to that of tRNA has suggested that the mode of action of RRF may be based on mimicry of tRNA. Directed hydroxyl radical probing ...

Directed hydroxyl radical probing of 16S ribosomal RNA in 70S ribosomes from internal positions of the RNA.

Published in Biochemistry

Directed hydroxyl radical probing of 16S ribosomal RNA from Fe(II) tethered to specific sites within the RNA was used to determine RNA-RNA proximities in 70S ribosomes. We have transcribed 16S ribosomal RNA in vitro as two separate fragments, covalently attached an Fe(II) probe to a 5 -guanosine-alpha-phosphorothioate at the junction between the tw...

Tagging ribosomal protein S7 allows rapid identification of mutants defective in assembly and function of 30 S subunits....

Published in Journal of Molecular Biology

Ribosomal protein S7 nucleates folding of the 16 S rRNA 3 major domain, which ultimately forms the head of the 30 S ribosomal subunit. Recent crystal structures indicate that S7 lies on the interface side of the 30 S subunit, near the tRNA binding sites of the ribosome. To map the functional surface of S7, we have tagged the protein with a Protein...

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

Published in RNA

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 ...

The 23 S rRNA environment of ribosomal protein L9 in the 50 S ribosomal subunit.

Published in Journal of Molecular Biology

Ribosomal protein L9 consists of two globular alpha/beta domains separated by a nine-turn alpha-helix. We examined the rRNA environment of L9 by chemical footprinting and directed hydroxyl radical probing. We reconstituted L9, or individual domains of L9, with L9-deficient 50 S subunits, or with deproteinized 23 S rRNA. A footprint was identified i...

Footprinting the sites of interaction of antibiotics with catalytic group I intron RNA.

Published in Science

Aminoglycoside inhibitors of translation have been shown previously to inhibit in vitro self-splicing by group I introns. Chemical probing of the phage T4-derived sunY intron shows that neomycin, streptomycin, and related antibiotics protected the N-7 position of G96, a universally conserved guanine in the binding site for the guanosine cofactor in...

Crystal structure of a translation termination complex formed with release factor RF2.

Published in Proceedings of the National Academy of Sciences

We report the crystal structure of a translation termination complex formed by the Thermus thermophilus 70S ribosome bound with release factor RF2, in response to a UAA stop codon, solved at 3 A resolution. The backbone of helix alpha5 and the side chain of serine of the conserved SPF motif of RF2 recognize U1 and A2 of the stop codon, respectively...

X-ray crystal structures of 70S ribosome functional complexes.

Published in Science

Structures of 70S ribosome complexes containing messenger RNA and transfer RNA (tRNA), or tRNA analogs, have been solved by x-ray crystallography at up to 7.8 angstrom resolution. Many details of the interactions between tRNA and the ribosome, and of the packing arrangements of ribosomal RNA (rRNA) helices in and between the ribosomal subunits, can...

Peptidyl transferase: protein, ribonucleoprotein, or RNA?

Published in Journal of Bacteriology

Rotation of the head of the 30S ribosomal subunit during mRNA translocation.

Published in Proceedings of the National Academy of Sciences

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 tra...