Chapter 22 : tRNA on the Ribosome: a Waggle Theory

Outline of ribosomal subunits, containing four empty tRNA sites, represented as dashed outlines. The 30S subunit is near the viewer, the SOS subunit more distant. Approximate locations for two ribosomal proteins (S21, LI) are shown at left, the path of the mRNA in the center, and the factor site containing EF-Tu is drawn at right

A simplified drawing of the tRNA tertiary structure. Positions of nucleotide changes observed to have significant effects on coding are numbered and marked by filled geometric shapes. Each type of symbol corresponds to a part of the text discussion.

Path of a single aa-tRNA through ribosomal E◀ P◀ A◀ T sites (right to upper left). Sites are represented by open outlines. Filled outlines contain tRNAs. Shading shows areas of prospective contact with the ribosomal RNAs. Open hatching represents tRNAs not necessarily in contact with rRNA; two different styles of hatching are used for two different tRNAs present simultaneously. Circles, squares, or triangles at the 3' end of tRNA sites symbolize amino acid sites (empty) or amino acids (filled). Dotted shield represents the ribosomal intermediate after peptide transfer and before translocation. Two hybrid tRNAs are explicitly shown. Double boxes represent measured rate constants of the steps depicted, at low temperatures. Boxes with two numbers show rates for cognate (top) and near-cognate (below) codon-anticodon interactions; three boxes with a third number (below line) also show the rate of comparable nucleic acid-nucleic acid interactions in solution, with no ribosome present, for comparison. For all steps but translocation, ribosomal rates are derived from quench-flow kinetics and attendant measurements on a tRNAPhe/poly(U) system at 5° by Thompson and collaborators (summarized in reference 120 ). However, the rate of cognate translocation was measured in stopped-flow fluorescence experiments on a similar system by Robertson et al. ( 95 ). Noncognate translocation (noncognate in the P site) was measured by Gast et al. ( 24 ); this value may not be comparable to the above because a less resolved experimental design was used, which required the intermediate formation of a peptide bond. Both apparent translocation rates are for saturating EF-G'GTP, recalculated for 5° using the measured activation energy. Oligonucleotide interaction rates come from Grosjean et al. ( 28 , 29 ).

Schematic diagram of some inter-tRNA distances in angstroms. Ribosomal E P A T sites are outlined with dotted lines. tRNAs within the sites are represented by open L shapes delimited by solid lines. Straight dashed lines and associated numbers indicate the approximate separation between fluors in angstroms. The dashed arc shows the approximate position of the 30S-50S subunit interface. Panel A shows distances in the E P/P state; panel B shows distances in the P/E A/P state. The distance in parentheses is from Fairclough and Cantor ( 18 ), and all others are taken from Paulsen et al. ( 80 ).

A convergent stereo pair (cross-eyed stereo that does not require a viewer) of the R and S tRNA configurations, displayed around a constant, central P-site tRNA. The illustration was created using Insight II (Ver 2.12, BioSym Inc.). Yeast tRNA coordinates are those of Jack et al. ( 40 ). tRNAs are represented by ribbons tracing the phosphodiester backbones, except that anticodon nucleotides and the substitution U33A in the P-site tRNA are explicitly indicated as stick structures emerging from the backbone ribbon. The short ribbon at the back is the message backbone; the upper (5′) UUC of the hexanucleotide UUCUUC was paired in standard A-form geometry to the P-site (central) anticodon, and A-form geometry is maintained for the lower (3′) three message nucleotides as a guide to interpretation.

Outline of an experiment to find mutations that enhance first-position wobble in an altered tRNATrp. Dotted boxes enclose the ambiguous positions generated by three mutagenic deoxynucleotides. The bracket at the left encloses all substitutions at positions 27–43; the nucleotides above the bar significantly enhanced first-position wobble. The nucleotides below the bar were not significantly different from the parental tRNA (C27•G43). The best mutant (GA) is significantly superior to those below, but the ordering within the other sequences does not always represent statistically justifiable superiority.

Energy (e) required for displacement (d) of a given size within a molecule: the curve represents e = kd2, where k is a force constant.