Modification and Sequence of tRNAs and mRNAs Impact Translational Speed and Accuracy: Insight into how Purines can Modulate Protein Synthesis

Abstract

The biochemical process of protein synthesis, known as translation, is comprised and dependent upon multiple types of RNA, including ribosomal RNA (rRNA), transfer RNA (tRNA), and messenger RNA (mRNA). While necessary, the importance of structure, sequence, and modification status of these different biomolecules is still being elucidated in the field. This body of work focuses on the roles that sequence and nucleotide modification status, in both tRNAs and mRNAs, have in impacting rates and efficiency of translation to better understand the roles of these biomolecules in peptide synthesis. I developed methodology used through this work, the reconstituted in vitro bacterial translation system, which I used to dissect translation down to its components at mechanistic, molecular, and atomic levels. There are known instances of ribosome pausing and slowed elongation rates, involved in protein and mRNA homeostasis, during translation of mRNAs encoding for polybasic peptides. I challenged the dogma that this process was caused by nascent peptide, decoupling and investigating role for individual contributions of mRNA sequence, peptide identity, and tRNA modification status in such synthesis (in the context of poly-lysine synthesis) and I determined that mRNA alone is sufficient to alter elongation rates of the ribosome in these instances. More strikingly, I discovered that when poly(A) is present within such mRNAs the ribosome can exhibit non-canonical translation. I defined a mechanism for this new-found process, deemed ribosome sliding, in which ribosome can lose frame and generating alternative protein products. My work found that this process, as well as other instances of canonical translation, can be perturbed with either the presence or absence of modifications to mRNA and tRNA species existing at the mRNA-tRNA interfaces within the ribosome. As such, my work serves a solid foundation for probing and investigating the process of translation and other biological process, which is necessary as the field considers the prevalence that RNA sequence and modification have in myriad disease states. To that end, my study of modifications for the purines adenosine (N6-methyladenosine [m6A]) and guanosine (N1-methylguanosine [m1G], N2-methylguanosine [m2G], and N2,N2-methylguanosine [m22G]) revealed that modifications to mRNA codons can drastically impact translation rates and efficiency in a position-dependent manner. I found that the presence of m6A in poly(A) relegates the extent at which the ribosome can slide on the message while guanosine studies suggest that the first two base-pairs between a codon and anticodon need at least 1 either N1 or N6 hydrogen bond for effective translation. As such results are also contingent upon the binding capacity of tRNAs, specifically its anticodon, I explored and summarized the known effects that anti-codon stem loop modifications of tRNA have on codon recognition and efficient translation as these concepts had been at a severe deficit in the field and is improved by my work. I specifically detail that ASL modifications are integral for frame maintenance, integral in -1 and +1 programmed ribosomal frameshift (PRF) events, and proper decoding and accommodation during translation. I also show that tRNA modifications are involved in stress-response situations, as is the case of antibiotic stress with hygromycin B, and can impact cross-talk intermolecular tRNA modification levels as well as translocation and cell viability. My work presented highlights the importance that mRNA and tRNA sequence and modification status has in modulating protein synthesis as well as a better understanding of how they do so.