In this project a number of peptidyl transferase antibiotics were studied, specifically a group of aminohexose cytosine nucleoside antibiotics and their interaction with a selected number of highly conserved ribonucleic acid (RNA) motifs, designed to represent their possible binding site within the ribosome. This group of antibiotics shows a wide range of interesting properties, including antiviral and anti-tumour activity, and as they bind to a particularly conserved region in the ribosome, they are likely to be difficult for microorganisms to develop resistance to. It is hoped that once the mechanism of action of these antibiotics is better understood, that modifications to the antibiotics can be effectively made to create new or hybrid antibiotics with more selective antibacterial, or indeed antiviral or anti-tumour properties. The nuclear magnetic resonance (NMR) structure of the RNA binding, peptidyl tranferase inhibitor antibiotics amicetin, blasticidin S and gougerotin, in their native solution states, have been successfully determined. The structures all exhibit a stable conformation, stabilised by intramolecular hydrogen bonds. Amicetin was observed to be folded, distinctly different from the linear, extended conformation of amicetin previously determined by X-ray crystallography. The structure of blasticidin S was found to be very similar to its X-ray crystal structure. Gougerotin was shown to form a similar conformation to blasticidin S, save that the end chain of gougerotin was bent at right angles to the rest of the molecule, forming a structure similar to that of the major bound X-ray crystal structure of blasticidin S. All the solution structures showed a similar conformation in the analogous regions of their chemical structure, suggesting that hybrid antibiotics could be produced.Two highly conserved RNA motifs of Halobacterium halobium (H. h.) and Escherichia coli (E. coli) 23S ribosomal RNAs were chosen to investigate their interaction with amicetin. The NMR structure of the H. h. and E. coli. 29-mer RNA motifs have been determined; the motifs both form well folded A-form RNA conformations. The E. coli NMR structure differs from the X-ray crystal structure of the motif contained within the ribosome, as a highly conserved adenine residue, which resides in a bulge strongly implicated with amicetin binding, folds into the helix as opposed to being flipped out. Instead, an adjacent cytosine residue partially flips out; whereas in the crystal structure, it is folded within the helix. The NMR stuctures of the H. h. motif differs from the X-ray crystal structure of the motif, contained within the ribosome, as none of the bases are flipped out and a number of non-canonical base pairs are formed in the solution structure. To continue this study, a fully 13C and 15N isotopically labelled version of the H. h. RNA sample has been partially assigned, and an initial structure determination has been performed, using ultra high field 1 GHz spectroscopy.Addition of amicetin to both the H. h. and E. coli 29-mer RNA samples were accompanied by discrete changes to the spectra, suggesting weak interaction between the two components. These can be qualitatively interpreted to changes induced in the local conformation of the RNA motifs and the amicetin arising from the formation of a complex, between the amicetin and the bulge region of the particular motif.

There is strong crystallographic evidence that the 23S rRNA is the only catalytic entity in the peptidyl transferase center. Various mechanisms for the catalysis of peptidyl transfer have been proposed. Recently, attention has been given to the idea that the 23S rRNA simply acts to position the tRNA for spontaneous peptidyl transfer and that chemical catalysis may play only a secondary role. Conserved nucleotides U2585 and U2506 are thought to be involved in positioning the 3’ ends of A- and P-site substrates based on the crystallographic evidence, and because mutagenesis at these sites severely impairs peptide bond formation. In this study, pure populations of ribosomes with either U2585A or U2506G mutations in the 23S rRNA were analyzed to test the hypothesis that substitutions at nucleotides U2585 and U2506 in the peptidyl transferase center impair peptide bond formation by altering the position of the 3’ end of P-site tRNA relative to the 23S rRNA. Pure populations of mutant or wild-type ribosomes were obtained by an affinity tagging system and probed with 32P-labeled [2N3A76]tRNAPhe to determine how the 3’ end of tRNA interacts with the ribosomal proteins and 23S RNA at the peptidyl transferase center. Some of the data for the ribosomes with a G at position 2506 are consistent with a model suggested by Schmeing and coworkers in which nucleotide U2506 breaks from its original wobble base pair with nucleotide G2583 during A-site tRNA binding and swings towards the 3’ end of P-site tRNA, while nucleotide U2585 simultaneously moves away from the 3’ end of P-site tRNA.

In eubacteria, ribosome stalling on broken messenger RNA transcripts can lead to cell death. The trans-translation quality control mechanism rescues many of these stalled ribosomes. In this process, tmRNA enters stalled ribosomes by mimicking a transfer RNA, accepting the stalled nascent peptide. The ribosome then releases the broken mRNA and resumes translation on a coding region within tmRNA itself. Translation of tmRNA marks the nascent peptide for destruction by the addition of a short proteolysis tag and the ribosome is released at a stop codon within the tmRNA open reading frame. An intriguing aspect of trans-translation is that the ribosome synthesizes one protein from two RNA templates. How is the proper site chosen on tmRNA to resume translation? Do the conserved pseudoknot structures help set the reading frame? Using a genetic selection to assay libraries of tmRNA mutants, we found that stable hairpin structures can functionally replace pseudoknot 1. We conclude that the role of pseudoknot 1 in tmRNA function is purely structural. Our results demonstrate that the inactivity of an RNA mutant designed to destroy a given structure should not be interpreted as proof that the structure is necessary for RNA function. Such mutations may only destabilize a global fold that could be formed equally well by an entirely different, stable structure. Broken mRNAs are not the only cause of ribosome stalling; stalling can also result from nascent peptide interactions with the ribosomal exit tunnel that inhibit peptidyl-transferase activity. SecM, TnaC, and ErmCL all stall ribosomes to regulate the expression of downstream genes. What other peptide sequences can cause ribosome stalling? We modified our tmRNA-based selection to screen libraries of random peptides and identified a number of novel stalling peptides, including the sequence FxxYxIWPP. This sequence interacts with the exit tunnel differently than SecM and TnaC as seen in studies using mutant ribosomes. Like SecM, stalling occurs on this sequence with the next aminoacyl tRNA trapped in the A site but unable to react with the nascent peptide. These results show that a variety of peptides can interact in the exit tunnel and peptidyl-transferase center to regulate ribosome activity.