Regulation of Protein Synthesis
Biological fitness is critically dependent upon the accurate flow of genetic information from DNA to RNA to protein. Breakdown in translational fidelity of the ribosome is detrimental to cells due its central role in the production of all proteins in every living organism. Major types of errors resulting from ribosome dysregulation include mRNA frame reading and tRNA selection errors. When the ribosome reads a non-three nucleotide codon, this causes expression of aberrant or nonsense proteins, which are then targeted for degradation. Recently, my laboratory has made exciting discoveries regarding the molecular basis for both these types of errors (Fagan et al., PNAS 2013; Fagan et al., RNA 2014; Maehigashi et al., PNAS 2014).
In collaboration with Prof. Kurt Fredrick at The Ohio State University, we determined that long-studied, single-nucleotide, “ribosome ambiguity” (ram) mutations in 16S rRNA promote conformational remodeling of important molecular bridges between the two ribosome subunits that allosterically regulate tRNA selection, allowing for tRNA miscoding (Fagan et al., PNAS 2013). Future experiments to extend these studies include determining how 16S rRNA ram mutations affect other aspects of elongation. Next, we determined the structural basis of ribosomal +1 frameshifting, i.e. changes in the mRNA reading frame that result from non-three-nucleotide reading of the genetic code (Fagan et al., RNA 2014; Maehigashi et al., PNAS 2014) (Figure 1). It had been presumed for decades that the three-nucleotide mRNA code was absolute. However, the discovery of suppressor tRNAs that decode a non-standard mRNA code indicated otherwise.
These tRNAs ‘suppressed’ an insertion in the mRNA sequence by reading greater or less than three nucleotides (‘frameshifting’), thus bringing the protein-coding region back into the correct frame. The discovery of “frameshift suppressor” tRNAs has stimulated the advent of the chemical biology age to develop tools for the direct incorporation of non-natural amino acids into recombinant proteins for downstream applications. Our long-term goal to develop a comprehensive model for mRNA frameshifting by the ribosome not only addresses an important fundamental question of the evolutionary origin of the three-nucleotide genetic code, but also promises to provide tools for its manipulation, expansion and thus reprogramming. Additional unexpected insights from these studies include the specific structural role that tRNA modifications play in fine-tuning interactions with the mRNA codon (Maehigashi et al., PNAS 2014). Future experiments will explore the roles that complex structured mRNAs play in maintenance of the three-nucleotide codon frame.
- Fagan CE, Dunkle JA, Maehigashi T, Dang MN, Deveraj A, Miles SJ, Qin D, Fredrick K and Dunham CM. (2013) Reorganization of an intersubunit bridge induced by disparate 16S ribosomal ambiguity mutations mimics an EF-Tu-bound state. Proc Natl Acad Sci 110(24):9716-21. PMCID: PMC3683721. (abstract)
- Maehigashi T*, Dunkle JA*, Miles SJ and Dunham CM. (2014) Structural insights into +1 frameshifting promoted by expanded or modification-deficient anticodon stem-loops. Proc Natl Acad Sci 111(35):12740-5. [*These authors contributed equally]. (abstract)
- Fagan CE, Maehigashi T, Dunkle JA, Miles SJ and Dunham CM. (2014) Structural insights into translational recoding by suppressor tRNASufJ. RNA 12:1944-55. PMCID: PMC4238358. (abstract)
- Washington A#, Benicewicz D#, Canzoneri J#, Fagan CE, Mwakwari S, Maehigashi T, Dunham CM* and Oyelere A*. (2014) Macrolide-Peptide Conjugates as Probes of the Path of Travel of the Nascent Peptides through the Ribosome. ACS Chemical Biology. 9(11):2621-31. PMCID: PMC4245169. [#These authors contributed equally; *Co-corresponding authors (abstract)
- Dunkle JA, Dunham CM (2015). Mechanisms of mRNA frame maintenance and its subversion during translation of the genetic code. Biochimie 114:90-6. PMCID: PMC4458409. (abstract)