Harry F. Noller

Gene organization and primary structure of a ribosomal RNA operon from Escherichia coli

The primary structure of the Escherichia coli rrnB ribosomal RNA operon has been determined. The sequence contains the genes for 16 S, 23 S and 5 S ribosomal RNAs, the secondary prophage site for phage λ, the transcriptional initiation and termination signals, as well as the spacers separating the various genetic elements. The entire sequence, ultimately derived from a 7508 base-pair BamHI fragment from the transducing phage λrifd18, is compared to homologous regions from other E. coli ribosomal RNA operons so far available. The main structural features of rRNA promoters and processing signals are shown to be conserved; rrnB has, however, two potential transcriptional termination sites, in contrast to rrnC. Comparison of the sequence of λrifd18 with sequences from other isolates of the rrB operon provides direct evidence for structural rearrangements within rRNA operons. Finally, two potential open reading frames for hitherto uncharacterized polypeptide chains have been found in the sequence closely flanking the rrnB operon.

The path of messenger RNA through the ribosome.

Using X-ray crystallography, we have directly observed the path of mRNA in the 70S ribosome in Fourier difference maps at 7 A resolution. About 30 nucleotides of the mRNA are wrapped in a groove that encircles the neck of the 30S subunit. The Shine-Dalgarno helix is bound in a large cleft between the head and the back of the platform. At the interface, only about eight nucleotides (-1 to +7), centered on the junction between the A and P codons, are exposed, and bond almost exclusively to 16S rRNA. The mRNA enters the ribosome around position +13 to +15, the location of downstream pseudoknots that stimulate -1 translational frame shifting.

Comparative Anatomy of 16-S-like Ribosomal RNA

Publisher Summary This chapter examines the range of the variation of secondary structure among the 16-S-like rRNAs. This brings into a larger structural context a recent detailed analysis of the individual helical elements and provides a basis for an accurate alignment of the corresponding regions of different primary structures. Computer-assisted comparative is used in the analysis of aligned sequences to describe the pattern of phylogenetic conservation for each nucleotide position in 16-S rRNA. A search for matching patterns among unpaired positions in the RNA chain then produces a list of candidates for potential base–base tertiary interactions. The completion of nucleotide sequences for 34 16-S-like rRNAs includes 4 eubacteria, 4 chloroplasts, 12 mitochondria, 4 archaebacteria, and 10 eukaryotes. Secondary structure models for these molecules have been developed in the course of refinement of the E. coli model, and have been used to arrive at improved sequence alignments for the 16-S-like rRNAs. Schematic drawings of (1) eubacterial, (2) archaebacterial, (3) eukaryotic cytoplasmic, (4) plant mitochondrial, (5) fungal mitochondrial, and (6) mammalian mitochondrial structures are shown in the chapter.

Crystal Structure of a 70S Ribosome-tRNA Complex Reveals Functional Interactions and Rearrangements

Our understanding of the mechanism of protein synthesis has undergone rapid progress in recent years as a result of low-resolution X-ray and cryo-EM structures of ribosome functional complexes and high-resolution structures of ribosomal subunits and vacant ribosomes. Here, we present the crystal structure of the Thermus thermophilus 70S ribosome containing a model mRNA and two tRNAs at 3.7 A resolution. Many structural details of the interactions between the ribosome, tRNA, and mRNA in the P and E sites and the ways in which tRNA structure is distorted by its interactions with the ribosome are seen. Differences between the conformations of vacant and tRNA-bound 70S ribosomes suggest an induced fit of the ribosome structure in response to tRNA binding, including significant changes in the peptidyl-transferase catalytic site.

Construction and fine mapping of recombinant plasmids containing the rrnB ribosomal RNA operon of E. coli.

Abstract We have constructed recombinant plasmids containing the entire Escherichia coli rrnB ribosomal RNA operon and segments thereof. Cloning of the 7.5-kb BamHI fragment, from λrifd18 which contains this operon, in plasmid vectors pBR 313 or pBR 322 is described. The 3.2-kb Eco RI Bam HI fragment containing the 3′ two-thirds of the 23 S rRNA gene, the 5S rRNA gene, and the terminator region has been cloned separately in pBR 313. As the nucleotide sequences of pBR 322 and the 7.5-kb fragment carrying the rrnB operon have been established, the entire 11.9-kb sequence of pKK 3535 is now known. This makes possible precise rearrangements and site-specific alterations of the ribosomal RNA operon; thus, pKK 3535 becomes a powerful tool for studies such as initiation and termination of transcription, processing of rRNA precursors, and investigations of the structure, function, and assembly of the ribosome itself. A detailed physical map of pKK 3535 is presented.

Interaction of elongation factors EF-G and EF-Tu with a conserved loop in 23S RNA.

The elongation factors EF-Tu and EF-G interact with ribosomes during protein synthesis1,2: EF-Tu presents incoming aminoacyl transfer RNA to the programmed ribosome as an EF-Tu-GTP-tRNA ternary complex and EF-G promotes translocation of peptidyl-tRNA and its associated messenger RNA from the A to the P site after peptidyl transfer. Both events are accompanied by ribosome-dependent GTP hydrolysis. Here we use chemical probes to investigate the possible interaction of these factors with ribosomal RNA in E. coli ribosomes. We observe EF-G-dependent footprints in vitro and in vivo around position 1,067 in domain II of 23S rRNA, and in the loop around position 2,660 in domain VI. EF-Tu gives an overlapping footprint in vitro at positions 2,655 and 2,661, but shows no effect at position 1,067. The 1,067 region is the site of interaction of the antibiotic thiostrepton2, which prevents formation of the EF-G–GTP–ribosome complex and is a site for interaction with the GTPase-related protein L11 (ref. 3). The universally conserved loop in the 2,660 region4 is the site of attack by the RNA-directed cytotoxins α-sarcin5 and ricin6, whose effects abolish translation and include the loss of elongation factor-dependent functions7 in eukaryotic ribosomes.

[33] Structural analysis of RNA using chemical and enzymatic probing monitored by primer extension

Publisher Summary This chapter focuses on the structural analysis of RNA using chemical and enzymatic probing monitored by primer extension. Chemical and enzymatic probing, monitored by primer extension, has become a powerful tool for the analysis of RNA structure. The reactivities of individual nucleotides composing large RNA molecules may be determined rapidly by utilizing a series of primers spaced at approximately 200 nucleotide intervals. In addition, numerous chemical reagents and nucleases may be employed as probes, since the only requirement is that they modify the template so as to produce pauses or stops in the progress of reverse transcriptase. The RNA, either alone or complexed with proteins and/or ligands, is incubated under suitable conditions with chemical or enzymatic probes. Dimethyl sulfate (DMS), kethoxal (KE) and l-cyclohexyl-3-(2-morpholinoethyl) carbodiimide metho- p -toluene sulfonate (CMCT) are employed for chemical probing, while ribonucleases A and T 1 , and V l nuclease are employed as enzymatic probes. The extent of the reactions is limited so that no more than a few stops are present within 300 nucleotide stretches in a given RNA molecule.

Following translation by single ribosomes one codon at a time

We have followed individual ribosomes as they translate single messenger RNA hairpins tethered by the ends to optical tweezers. Here we reveal that translation occurs through successive translocation-and-pause cycles. The distribution of pause lengths, with a median of 2.8 s, indicates that at least two rate-determining processes control each pause. Each translocation step measures three bases—one codon—and occurs in less than 0.1 s. Analysis of the times required for translocation reveals, surprisingly, that there are three substeps in each step. Pause lengths, and thus the overall rate of translation, depend on the secondary structure of the mRNA; the applied force destabilizes secondary structure and decreases pause durations, but does not affect translocation times. Translocation and RNA unwinding are strictly coupled ribosomal functions.

mRNA Helicase Activity of the Ribosome

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 translation system made up of purified components, we show that ribosomes are able to disrupt downstream helices, including a perfect 27 base pair helix of predicted T(m) = 70 degrees . Using helices of different lengths and registers, the helicase active site can be localized to the middle of the downstream tunnel, between the head and shoulder of the 30S subunit. Mutation of residues in proteins S3 and S4 that line the entry to the tunnel impairs helicase activity. We conclude that the ribosome itself is an mRNA helicase and that proteins S3 and S4 may play a role in its processivity.

Chloramphenicol, erythromycin, carbomycin and vernamycin B protect overlapping sites in the peptidyl transferase region of 23S ribosomal RNA.

Using dimethyl sulfate and kethoxal, we have probed antibiotic-ribosome complexes, and identified sites of interaction of chloramphenicol, erythromycin, carbomycin, vernamycin B and viomycin with 23S rRNA. Chloramphenicol, erythromycin, carbomycin and vernamycin B protect overlapping nonequivalent sites in the central loop of domain V. From the known functional effects of these drugs and their protection patterns, we infer that peptidyl transferase is inhibited as a result of binding antibiotics proximal to A-2451, whereas antibiotics bound proximal to A-2058 interfere with growth of the nascent polypeptide chain. Vernamycin B also strongly protects A-752, implying that this region of domain II is proximal to the central loop of domain V. Viomycin, which affects translocation and subunit dissociation, protects U-913 and G-914.