Ribosomes are universally conserved macromolecular machines found within all living cells that catalyze protein synthesis, one of nature’s most fundamental processes. Ribosomes synthesize proteins, which are polymeric chains of amino acids, by incorporating the amino acids one at a time via aminoacylated-transfer RNAs (aa-tRNAs), based on translation of the sequence of triplet- nucleotide codons presented by the messenger RNA (mRNA) template that is a direct readout of genomic DNA. Recent biochemical, structural, dynamic, and computational studies have uncovered large-scale conformational changes of the ribosome, its tRNA substrates, and the additional protein translation factors that play important roles in regulating protein synthesis, especially during the elongation phase of translation when the bulk of each protein is synthesized. How the ribosome, its translation elongation factors, tRNAs, and mRNA physically coordinate and regulate the movements of the tRNAs carrying amino acids into, through, and out of the ribosome remains one of the more fundamental questions in the mechanistic studies of protein synthesis. A complete understanding of the conformational dynamics of ribosomal complexes will improve our knowledge of how translation is regulated, including how ribosome-targeting antibiotics regulate translation elongation, and will provide crucial information for designing next-generation antibiotics. In this thesis I have investigated the conformational dynamics of the ribosome during the elongation phase of protein synthesis at the single-molecule level using single-molecule fluorescence resonance energy transfer (smFRET) microscopy experiments. Specifically, I have studied ribosomal dynamics during the elongation phase of translation in the presence of a tRNAPro in the context of an mRNA that has the propensity to shift out of the reading frame. My studies have revealed information about the mechanistic and regulatory functions of the posttranscriptional modifications of tRNAPro in a context in which the ribosomal complex has the propensity to undergo non-programmed +1-frameshifting, in which the tRNA-mRNA base pairing shifts one base toward the 3’ end of the mRNA, and if unchecked, leads to the synthesis of a polypeptide with a completely different sequence of amino acids. My data suggests that in this context, the mechanism underlying non-programmed +1-frameshifting involves the tRNA shifting out of frame prior to the tRNA being accommodated in the P site, i.e. either while the tRNA is in the A site, or more likely, during translocation of the tRNA from the A site to the P site, and not while the tRNA is already occupying the P site, as previously proposed.

The human immunodeficiency virus type 1 (HIV-1) uses a mechanism of genetic recoding known as programmed ribosomal frameshifting to translate the proteins encoded by the pol gene. The pol gene overlaps the preceding gag gene in the -1 reading frame relative to gag. It contains neither a start codon nor an internal ribosome entry site (IRES) to initiate translation of its proteins. Rather the host ribosomes are forced to pause due to tension placed on the mRNA when they encounter a specific secondary structural element in the mRNA. This tension is relieved by disruption of the contacts between the mRNA codons and tRNA anticodons at a �slippery� sequence within the ribosomal decoding centre. Re-pairing of the tRNAs occurs in the new -1 frame after movement of the mRNA backwards by one nucleotide, allowing the ribosome to translate the pol gene as a Gag-Pol polyprotein. A change in ratio of Gag to Gag-Pol proteins affects viral assembly, and most significantly dramatically reduces viral infectivity. The prevailing model for the mechanism of -1 frameshifting has focussed on a pre-translocational event, where slippage occurs when the slippery sequence is within the ribosomal A and P sites. This model precludes a contribution from the codon immediately downstream of the slippery sequence leading into the secondary structural element. I have termed this the �intercodon�. Often at frameshifting sites it is a termination codon, whereas in HIV-1 it is a glycine codon, GGG. When the intercodon within the frameshift element was changed from the wild-type GGG to a termination codon UGA, the efficiency of frameshifting decreased 3-4-fold in an in vivo assay in cultured human cells. This result mimicked previous data in the group within bacterial cells and cultured monkey COS-7 cells. Changing the first nucleotide of the intercodon to each of the three other bases altered frameshifting to varying degrees, but not following expected patterns for base stacking effects. Such a result would support a post-translocational model for -1 frameshifting. It suggested that the intercodon might be within the ribosomal A site before frameshifting, and that the slippery sequence was therefore within the P and E sites. This was investigated by modulating the expression of decoding factors for the intercodon - the release factor eRF1 and cognate suppressor tRNAs when it was either of the UGA or UAG termination codons, and tRNA[Gly] for the native GGG glycine codon. These were predicted to affect frameshifting only if slippage were occurring when the ribosomal elongation cycle was in the post-translocational state. Overexpression of tRNA[Gly] gave inconsistent effects on frameshifting in vivo, implying that its concentration may not be limiting within the cell. When eRF1 was overexpressed or depleted by RNAi, significant functional effects of decreased or increased stop codon readthrough respectively were documented. Expression of suppressor tRNAs increased readthrough markedly in a stop codon-specific manner. These altered levels of eRF1 expression were able to modulate the +1 frameshifting efficiency of the human antizyme gene. Overexpression of eRF1 caused significant reduction of frameshifting of the HIV-1 element with the UAG or UGA intercodon. Depletion of the protein by contrast had unexplained global effects on HIV-1 frameshifting. Suppressor tRNAs increased frameshifting efficiency at the UAG or UGA specifically in a cognate manner. These results strongly indicate that a post-translocational mechanism of frameshifting is used to translate the HIV-1 Gag-Pol protein. A new model (�almost� post-translocational) has been proposed with -1 frameshifting occurring for 1 in 10 or 20 ribosomal passages during the end stages of translocation, because of opposing forces generated by translocation and by resistance to unwinding of the secondary structural element. With translocation still incomplete the slippery sequence is partially within the E and P sites, and the intercodon partially within the A site. The nature of the intercodon influences frameshifting efficiency because of how effectively the particular decoding factor is able to bind to the partially translocated intercodon and maintain the normal reading frame.

No description available.

No description available.

No description available.

Mechanical unfolding of -1 frameshift signals such as RNA pseudoknots have aimed to test the hypothesis that the stability of the pseudoknot is directly correlated to the frameshifting efficiency. Here we report unfolding of the Beet Western Yellow Virus (BWYV) pseudoknot by optical tweezers experiments complemented by computer simulations using steered molecular dynamics (SMD). Seven pseudoknot scenarios were studied: the wild-type pseudoknot in the presence and absence of Mg<super>2+</super>, the wild-type pseudoknot at high pH (deprotonated C8), and C8U, C8A, A24G, G19U, and G19UC mutant constructs. The mutants were selected to probe three key structural features of the BWYV pseudoknot, a triple-stranded helix at the base of stem 1, the stem junction region of stem 1 and stem 2, and a unique quadruple base-pair interaction involving a protonated cytosine in position 8 (C8). These regions are thought to control ribosomal frameshifting by different strategies such as thermodynamic stability, kinetic influences, and dynamics involving contacts with the ribosome. In addition, the mutants have been shown to either abolish frameshifting ability of the pseudoknot (C8 mutant cases and A24G), or actually increase the frameshifting efficiency (as seen with G19U and G19UC). We find three major conclusions from the stretching of the pseudoknot constructs with optical tweezers. First, stretching in the absence of Mg<super>2+</super> results in no observed unfolding transitions. We interpret this to mean that magnesium is indispensible for the stable folding of the pseudoknot. Second, we found that frameshifting efficiency is not correlated with the force required to unfold the pseudoknots. However, we observe the unfolding of stem 1 in all of the pseudoknots stretched, where stem 2 unfolding is below our noise level. For this reason, we cannot rule out the possibility that an estimate of the thermodynamic stability of the entire pseudoknot would correlate with frameshifting efficiency. And third, we found that each pseudoknot mutant that resulted in reduced frameshifting efficiency also exhibited more off-equilibrium unfolding transitions that the wild-type pseudoknot under comparable loading rates. We conclude from these studies that the resistance of a pseudoknot to unfolding is controlled by both thermodynamic and kinetic parameters. We then suggest new technologies that would allow for greater resolution in order to correlate pseudoknot unfolding behavior with -1 programmed ribosomal frameshifting events.

No description available.

The human immunodeficiency virus type 1 (HIV-1) uses a mechanism of genetic recoding known as programmed ribosomal frameshifting to translate the proteins encoded by the pol gene. The pol gene overlaps the preceding gag gene in the -1 reading frame relative to gag. It contains neither a start codon nor an internal ribosome entry site (IRES) to initiate translation of its proteins. Rather the host ribosomes are forced to pause due to tension placed on the mRNA when they encounter a specific secondary structural element in the mRNA. This tension is relieved by disruption of the contacts between the mRNA codons and tRNA anticodons at a �slippery� sequence within the ribosomal decoding centre. Re-pairing of the tRNAs occurs in the new -1 frame after movement of the mRNA backwards by one nucleotide, allowing the ribosome to translate the pol gene as a Gag-Pol polyprotein. A change in ratio of Gag to Gag-Pol proteins affects viral assembly, and most significantly dramatically reduces viral infectivity. The prevailing model for the mechanism of -1 frameshifting has focussed on a pre-translocational event, where slippage occurs when the slippery sequence is within the ribosomal A and P sites. This model precludes a contribution from the codon immediately downstream of the slippery sequence leading into the secondary structural element. I have termed this the �intercodon�. Often at frameshifting sites it is a termination codon, whereas in HIV-1 it is a glycine codon, GGG. When the intercodon within the frameshift element was changed from the wild-type GGG to a termination codon UGA, the efficiency of frameshifting decreased 3-4-fold in an in vivo assay in cultured human cells. This result mimicked previous data in the group within bacterial cells and cultured monkey COS-7 cells. Changing the first nucleotide of the intercodon to each of the three other bases altered frameshifting to varying degrees, but not following expected patterns for base stacking effects. Such a result would support a post-translocational model for -1 frameshifting. It suggested that the intercodon might be within the ribosomal A site before frameshifting, and that the slippery sequence was therefore within the P and E sites. This was investigated by modulating the expression of decoding factors for the intercodon - the release factor eRF1 and cognate suppressor tRNAs when it was either of the UGA or UAG termination codons, and tRNA[Gly] for the native GGG glycine codon. These were predicted to affect frameshifting only if slippage were occurring when the ribosomal elongation cycle was in the post-translocational state. Overexpression of tRNA[Gly] gave inconsistent effects on frameshifting in vivo, implying that its concentration may not be limiting within the cell. When eRF1 was overexpressed or depleted by RNAi, significant functional effects of decreased or increased stop codon readthrough respectively were documented. Expression of suppressor tRNAs increased readthrough markedly in a stop codon-specific manner. These altered levels of eRF1 expression were able to modulate the +1 frameshifting efficiency of the human antizyme gene. Overexpression of eRF1 caused significant reduction of frameshifting of the HIV-1 element with the UAG or UGA intercodon. Depletion of the protein by contrast had unexplained global effects on HIV-1 frameshifting. Suppressor tRNAs increased frameshifting efficiency at the UAG or UGA specifically in a cognate manner. These results strongly indicate that a post-translocational mechanism of frameshifting is used to translate the HIV-1 Gag-Pol protein. A new model (�almost� post-translocational) has been proposed with -1 frameshifting occurring for 1 in 10 or 20 ribosomal passages during the end stages of translocation, because of opposing forces generated by translocation and by resistance to unwinding of the secondary structural element. With translocation still incomplete the slippery sequence is partially within the E and P sites, and the intercodon partially within the A site. The nature of the intercodon influences frameshifting efficiency because of how effectively the particular decoding factor is able to bind to the partially translocated intercodon and maintain the normal reading frame.

Thesis (Ph.D.)--University of Wisconsin--Madison, 2006. / Includes bibliographical references (p. 405-431). Also available on the Internet.

Thesis (M.S.) -- University of Maryland, College Park, 2006. / Thesis research directed by: Dept. of Cell Biology & Molecular Genetics. Title from t.p. of PDF. Includes bibliographical references. Published by UMI Dissertation Services, Ann Arbor, Mich. Also available in paper.