Showing papers by "Harry F. Noller published in 1997"

Ribosomes and translation

Abstract: The ribosome is a large multifunctional complex composed of both RNA and proteins. Biophysical methods are yielding low-resolution structures of the overall architecture of ribosomes, and high-resolution structures of individual proteins and segments of rRNA. Accumulating evidence suggests that the ribosomal RNAs play central roles in the critical ribosomal functions of tRNA selection and binding, translocation, and peptidyl transferase. Biochemical and genetic approaches have identified specific functional interactions involving conserved nucleotides in 16S and 23S rRNA. The results obtained by these quite different approaches have begun to converge and promise to yield an unprecedented view of the mechanism of translation in the coming years.

Mapping the inside of the ribosome with an RNA helical ruler

Abstract: The structure of ribosomal RNA (rRNA) in the ribosome was probed with hydroxyl radicals generated locally from iron(II) tethered to the 5′ ends of anticodon stem-loop analogs (ASLs) of transfer RNA The ASLs, ranging in length from 4 to 33 base pairs, bound to the ribosome in a messenger RNA–dependent manner and directed cleavage to specific regions of the 16S, 23S, and 5S rRNA chains The positions and intensities of cleavage depended on whether the ASLs were bound to the ribosomal A or P site, and on the lengths of their stems These data predict the three-dimensional locations of the rRNA targets relative to the positions of A- and P- site transfer RNAs inside the ribosome

Identification of 2-hydroxyl groups required for interaction of a tRNA anticodon stem-loop region with the ribosome

Abstract: Synthetic RNA stem loops corresponding to positions 28-42 in the anticodon region of tRNA(Phe) bind efficiently in an mRNA-dependent manner to ribosomes, whereas those made from DNA do not. In order to identify the positions where ribose is required, the anticodon stem-loop region of tRNA(Phe) (Escherichia coli) was synthesized chemically using a mixture of 2'-hydroxyl- and 2'-deoxynucleotide phosphoramidites. Oligonucleotides whose ribose composition allowed binding were retained selectively on nitrocellulose filters via binding to 30S ribosomal subunits. The binding-competent oligonucleotides were submitted to partial alkaline hydrolysis to identify the positions that were enriched for ribose. Quantification revealed a strong preference for a 2'-hydroxyl group at position U33. This was shown directly by the 50-fold lower binding affinity of a stem loop containing a single deoxyribose at position U33. Similarly, defective binding of the corresponding U33-2'-O-methyl-substituted stem-loop RNA suggests that absence of the 2'-hydroxyl group, rather than an altered sugar pucker, is responsible. Stem-loop oligoribonucleotides from different tRNAs with U33-deoxy substitutions showed similar, although quantitatively different effects, suggesting that intramolecular rather than tRNA-ribosome interactions are affected. Because the 2'-hydroxyl group of U33 was shown to be a major determinant of the U-turn of the anticodon loop in the crystal structure of tRNA(Phe) in yeast, our finding might indicate that the U-turn conformation in the anticodon loop is required and/or maintained when the tRNA is bound to the ribosomal P site.

Structure of a conserved RNA component of the peptidyl transferase centre

Abstract: The structure of a conserved hairpin loop involved in peptidyl-tRNA recognition by 50S ribosomal subunits has been solved by NMR. The loop is closed by a novel G-C base pair and presents guanine residues for RNA recognition.