Translational control and regulation
Protein synthesis is a complex and highly regulated process coordinated by the large macromolecular machine, the ribosome. The ribosome is a one of the most conserved and critical macromolecular complexes and over four decades of biochemical, genetic and structural analyses have provided immense insights into its function. The Dunham laboratory is interested in determining a molecular framework for how dysregulation of the bacterial ribosome results from different cellular environments that control bacterial proliferation and persistence.
Specific projects in the lab include: 1) determination of the molecular mechanisms for mRNA frameshifting that cause the ribosome to read an alternative genetic code resulting in different gene expression patterns; 2) the structural and functional basis of negative regulation of their own expression by toxin-antitoxin complexes during steady state growth, and the rapid translation of specific mRNAs to change the cellular proteome during stress conditions; 3) the role of ribosomal RNA modification enzymes that confer antibiotic resistance.
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)
Toxin-mediated degradation of mRNA during the bacterial stringent response
Bacteria adapt to stressful conditions by rapidly adjusting their metabolic rates via global regulatory responses. General mechanisms for adaption include the SOS response, general stress response, the heat-shock response and the stringent response. Factors involved in the stringent response have been identified across diverse microorganisms but their detailed molecular mechanisms of target recognition and action are still unknown. One major focus of my lab is to study how specific proteins involved in the stringent response repress translation, allowing bacteria to enter a nonreplicative latent state known as persistence.
The stringent response is one of the most important regulatory circuits in bacteria. The accumulation of (p)ppGpp modifies global cellular metabolism in response to changing micro-environments to optimize growth, and ultimately bacterial survival, in a very short time frame (Figure 2). Toxin-antitoxin (TA) gene pairs facilitate cell survival during the stringent response and have been implicated in biofilm formation, persistence during antibiotic treatment and bacterial pathogenesis. During times of stress such as nutritional deprivation, TA systems fine tune basic cellular processes for survival. One major function of toxin proteins is to halt protein synthesis, which conserves energy for cell survival and additionally produces truncated proteins for degradation and replenishment of the amino acid pool. Repression of translation by toxin proteins typically occurs by degradation of mRNA bound to a translating ribosome in a codon-dependent manner. This precision implies a sophisticated mechanism of RNA recognition similar to how tRNAs decode mRNA codons.
The Dunham laboratory has been studying two different ribosome-dependent toxin-antitoxin systems: Proteus vulgaris HigBA and E. coli DinJ-YafQ (Figure 3). The structures of the transrepressor complexes revealed that manner in which each toxin YafQ and HigB is repressed and furthermore that each contains distinct DNA-binding motifs implying novel repression. Future experiments aimed at determining how they specifically recognize both their RNA and DNA targets are currently ongoing.
- Schureck MA, Maehigashi T, Miles SJ, Marquez J, Ei Cho S, Erdman R and Dunham CM. (2014) Structure of the P. vulgaris HigB-(HigA)2-HigB toxin-antitoxin complex. Journal of Biological Chemistry 289(2):1060-70. PMCID: PMC3887174. (abstract)
- Cruz JW, Rothenbacher FP, Maehigashi T, Lane WS, Dunham CM, Woychik NA. (2014) Doc toxin is a kinase that inactivates elongation factor Tu. Journal of Biological Chemistry 289(11):7788-99. PMCID: PMC3953291. (abstract)
- Ruangprasert A*, Maehigashi T*, Miles SJ, Giridharan N, Liu JX and Dunham CM. (2014) Mechanisms of toxin inhibition and transcriptional repression by E. coli DinJ-YafQ. Journal of Biological Chemistry 289(30):20559-69. PMCID: PMC4110269. [*These authors contributed equally]. (abstract)
- Cruz JW, Sharp JD, Hoffer ED, Maehigashi T, Vyedenskaya IO, Konkimalla A, Husson RN, Nickels BE, Dunham CM and Woychik NA (2015). Growth-regulating Mycobacterium tuberculosis VapC-mt4 toxin is an isoacceptor-specific tRNase. Nature Communications 6:7480. PMID: 26158745. (abstract).
- Maehigashi T*, Ruangprasert A*, Miles SJ and Dunham CM (2015). Molecular basis of ribosome regulation and mRNA hydrolysis by the E. coli YafQ toxin. Nucleic Acids Research epub Aug 10. PMID: 26261214. *These authors contributed equally. (abstract).
- Schureck MA and Dunham CM. (2014) Bacterial warfare again targets the ribosome. Structure 22(5):661-2. PMID: 24807074. (PMCID in process) (abstract)
RNA modification and antibiotic resistance
Increasing global spread of antibiotic resistance among pathogenic bacteria threatens a post-antibiotic era in healthcare. Detailed studies of resistance mechanisms are therefore urgently required. The ribosome is a major antibiotic target, but bacteria can acquire resistance by modification of drug-binding sites. In collaboration with Prof. Graeme Conn’s lab at Emory University, we have been studying the molecular basis for antibiotic-resistance arising via enzymatic modification of the small ribosomal subunit. We solved the X-ray structure of the first molecular ‘snapshot’ of 30S recognition by a human pathogen-derived, aminoglycoside-resistance rRNA methyltransferase, NpmA (Dunkle et al., PNAS 2014).
Future experiments include determining how structurally dissimilar but functionally equivalent methyltransferases recognize the 30S ribosomal subunit. Our initial studies indicate that subtle differences in methyltransferase residues that direct recognition of 30S have a direct result in whether the enzyme is preloaded with its obligate SAM cofactor or whether the ribosome plays a role in recycling the reaction by-product SAH for SAM while the methyltransferase is bound to the ribosome.
- Dunkle JA, Vinnal K, Desai PM, Zelinskaya N, Savic M, West DM, Conn GL* and Dunham CM*. (2014) Molecular recognition and modification of the 30S ribosome by the aminoglycoside-resistance methyltransferase NpmA. Proc Natl Acad Sci 111(17):6275-80. PMCID: PMC4035980. [*Co-corresponding authors] (abstract)