I read this book the same weekend that the Packers took on the Rams, and the experience of the latter event, obviously, colored my judgment. Although I abhor anything that smacks of being a handbook (like, \"How to Earn a Merit Badge in Neurosurgery\") because too many volumes in biomedical science already evince a boyscout-like approach, I must confess that parts of this volume are fast, scholarly, and significant, with certain reservations. I like parts of this well-illustrated book because Dr. Sj6strand, without so stating, develops certain subjects on technique in relation to the acquisition of judgment and sophistication. And this is important! So, given that the author (like all of us) is somewhat deficient in some areas, and biased in others, the book is still valuable if the uninitiated reader swallows it in a general fashion, realizing full well that what will be required from the reader is a modulation to fit his vision, propreception, adaptation and response, and the kind of problem he is undertaking. A major deficiency of this book is revealed by comparison of its use of physics and of chemistry to provide understanding and background for the application of high resolution electron microscopy to problems in biology. Since the volume is keyed to high resolution electron microscopy, which is a sophisticated form of structural analysis, but really morphology in a modern guise, the physical and mechanical background of The instrument and its ancillary tools are simply and well presented. The potential use of chemical or cytochemical information as it relates to biological fine structure , however, is quite deficient. I wonder when even sophisticated morphol-ogists will consider fixation a reaction and not a technique; only then will the fundamentals become self-evident and predictable and this sine qua flon will become less mystical. Staining reactions (the most inadequate chapter) ought to be something more than a technique to selectively enhance contrast of morphological elements; it ought to give the structural addresses of some of the chemical residents of cell components. Is it pertinent that auto-radiography gets singled out for more complete coverage than other significant aspects of cytochemistry by a high resolution microscopist, when it has a built-in minimal error of 1,000 A in standard practice? I don't mean to blind-side (in strict football terminology) Dr. Sj6strand's efforts for what is \"routinely used in our laboratory\"; what is done is usually well done. It's just that …

Methods in Enzymology.

5.1. Binding Sites of the Aminoglycosides 1185 5.2. Importance of Electrostatic Interactions 1185 5.3. Nonionic Interactions 1185 5.4. Pseudo-Base Pair Interactions 1186 5.5. Water-Mediated Contacts 1186 5.6. Shape Complementarity and Conformational Adaptation 1186 6. Assays for Evaluating RNA Binding 1186 6.1. Methods Utilizing Fluorescently Labeled RNA 1186 6.2. NMR-Based Methods 1187 6.3. Electrospray Ionization Mass Spectrometry-Based Methods 1187

Targeting RNA with small molecules

▪ Abstract The underlying basis for the accuracy of protein synthesis has been the subject of over four decades of investigation. Recent biochemical and structural data make it possible to understand at least in outline the structural basis for tRNA selection, in which codon recognition by cognate tRNA results in the hydrolysis of GTP by EF-Tu over 75 A away. The ribosome recognizes the geometry of codon-anticodon base pairing at the first two positions but monitors the third, or wobble position, less stringently. Part of the additional binding energy of cognate tRNA is used to induce conformational changes in the ribosome that stabilize a transition state for GTP hydrolysis by EF-Tu and subsequently result in accelerated accommodation of tRNA into the peptidyl transferase center. The transition state for GTP hydrolysis is characterized, among other things, by a distorted tRNA. This picture explains a large body of data on the effect of antibiotics and mutations on translational fidelity. However, many fu...

https://www.annualreviews.org/doi/pdf/10.1146/annurev.biochem.74.061903.155440

Structural insights into translational fidelity

Using single-molecule fluorescence spectroscopy, time-resolved conformational changes between fluorescently labeled tRNA have been characterized within surface-immobilized ribosomes proceeding through a complete cycle of translation elongation. Fluorescence resonance energy transfer was used to observe aminoacyl-tRNA (aa-tRNA) stably accommodating into the aminoacyl site (A site) of the ribosome via a multistep, elongation factor-Tu dependent process. Subsequently, tRNA molecules, bound at the peptidyl site and A site, fluctuate between two configurations assigned as classical and hybrid states. The lifetime of classical and hybrid states, measured for complexes carrying aa-tRNA and peptidyl-tRNA at the A site, shows that peptide bond formation decreases the lifetime of the classical-state tRNA configuration by approximately 6-fold. These data suggest that the growing peptide chain plays a role in modulating fluctuations between hybrid and classical states. Single-molecule fluorescence resonance energy transfer was also used to observe aa-tRNA accommodation coupled with elongation factor G-mediated translocation. Dynamic rearrangements in tRNA configuration are also observed subsequent to the translocation reaction. This work underscores the importance of dynamics in ribosome function and demonstrates single-particle enzymology in a system of more than two components.

/pdf/trna-dynamics-on-the-ribosome-during-translation-1s4x58h0e1.pdf

tRNA dynamics on the ribosome during translation

Transfer RNA shields specific nucleotides in 16S ribosomal RNA from attack by chemical probes

The anticodon loop of tRNA contains a number of conserved or semiconserved nucleotides. In most tRNAs, a highly modified purine is found at position 37 immediately 3′ to the anticodon. Here, we examined the role of the base at position 37 for tRNAPhe binding to the A site of Escherichia coli ribosomes. Affinities and rate constants of A-site binding of native yeast peptidyl-tRNAPhe with hypermodified G (wybutine), or of unmodified peptidyl-tRNAPhe transcripts with G, A, C, or U, at position 37 were measured. The data indicate that purines stabilize binding due to stronger stacking and additional interactions with the ribosome mediated by Mg2+ ions. Paromomycin, an antibiotic that binds to 16S rRNA in the decoding center, greatly stabilized tRNAs in the A site and abolished the Mg2+-dependence of binding. Comparison of binding enthalpies and entropies suggests that hypermodification of the base at position 37 does not affect stacking in the codon–anticodon complex, but rather decreases the entropic penalty for A-site binding. Substitution of purines with pyrimidines at position 37 increases the rates of tRNA binding to and dissociation from the A site. The data suggest that initial binding of tRNA to the A site is followed by a rate-limiting rearrangement of the anticodon loop or the ribosome decoding center that is favored by purines at position 37 and involves stronger stacking, additional Mg2+ binding, and interactions with 16S rRNA.

/pdf/purine-bases-at-position-37-of-trna-stabilize-codon-1lu3yzgc6k.pdf

Purine bases at position 37 of tRNA stabilize codon-anticodon interaction in the ribosomal A site by stacking and Mg2+-dependent interactions.

30S subunits were isolated capable to bind simultaneously two molecules of Phe-tRNAPhe (or N-Acetyl-Phe-tRNAPhe), both poly(U) dependent. The site with higher affinity to tRNA was identified as P site. tRNA binding to this site was not inhibited by low concentrations of tetracycline (2 x 10(-5)M) and, on the other hand, N-Acetyl-Phe-tRNAPhe, initially prebound to the 30S.poly(U) complex in the presence of tetracycline, reacted with puromycin quantitatively after addition of 50S subunits. The site with lower affinity to tRNA revealed features of the A site: tetracycline fully inhibited the binding of both Phe-tRNAPhe and N-Acetyl-Phe-tRNAPhe. Binding of two molecules of Phe-tRNAPhe to the 30S.poly(U) complex followed by the addition of 50S subunits resulted in the formation of (Phe)2-tRNAPhe in 75-90% of the reassociated 70S ribosomes. These results prove that isolated 30S subunits contain two physically distinct centers for the binding of specific aminoacyl- (or peptidyl-) tRNA. Addition of 50S subunits results in the formation of whole 70S ribosomes with usual donor and acceptor sites.

Mechanism of codon-anticodon interaction in ribosomes. Direct functional evidence that isolated 30S subunits contain two Codon-specific binding sites for transfer RNA

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1016/0014-5793%2892%2980380-Y

Puromycin reaction for the A site-bound peptidyl-tRNA.

Purpuromycin, an antibiotic produced by Actinoplanes ianthinogenes, had been reported previously to inhibit protein synthesis. In the present report, we demonstrate that the mechanism of action of this antibiotic is quite novel in that it binds with fairly high affinity to all tRNAs, inhibiting their acceptor capacity. Although more than one molecule of purpuromycin is bound to each tRNA molecule, the inhibitory activity of this antibiotic was found to be selective for the tRNA acceptor function; in fact, after the aminoacylation step, purpuromycin was found to affect none of the other tested functions of tRNA (interaction with the ribosomal P- and A-sites and interaction with translation factors). Accordingly, purpuromycin was found to inhibit protein synthesis only when translation depended on the aminoacylation of tRNA and not when the system was supplemented with pre-formed aminoacyl-tRNAs. Because purpuromycin did not interfere with the ATP-PPi exchange reaction of the synthetase or with the initial interaction of the enzyme with its tRNA substrate, the basis for the inhibition of aminoacylation is presumably the formation of a nonproductive synthetase-tRNA complex in the presence of purpuromycin in which the tRNA is unable to be charged with the corresponding amino acid.

Purpuromycin: an antibiotic inhibiting tRNA aminoacylation.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1016/0014-5793%2880%2980302-4

Makhno Vi

Papers

Purine bases at position 37 of tRNA stabilize codon-anticodon interaction in the ribosomal A site by stacking and Mg2+-dependent interactions.

Mechanism of codon-anticodon interaction in ribosomes. Direct functional evidence that isolated 30S subunits contain two Codon-specific binding sites for transfer RNA

Puromycin reaction for the A site-bound peptidyl-tRNA.

Purpuromycin: an antibiotic inhibiting tRNA aminoacylation.

MECHANISM OF CODON-ANTICODON INTERACTION IN RIBOSOMES Codon-anticodon interaction of aminoacyl-tRNA at the ribosomal donor site