One of our objectives is to understand how tRNAs work in protein synthesis. Initiation is the step of much of translational regulation of mRNAs. It is also often the rate limiting step in protein synthesis. Therefore, a major part of our effort is focused on initiator tRNAs, which function exclusively in initiation of protein synthesis and possess many properties different from those of other tRNAs. Our goals are:
- to identify the molecular mechanisms by which the various components of the translational machinery such as the formylating enzyme, the initiation factors, the elongation factor, the ribosome, etc. distinguish initiator tRNAs from other tRNAs in the cell; and
- to analyze the function of the initiator tRNA in initiation of protein synthesis.
Initiation of Protein Synthesis in E. coli: We showed that mutant initiator tRNAs with changes in the anticodon sequence can initiate protein synthesis from an initiation codon that is complementary to the tRNA anticodon and with amino acids other than methionine. This finding made possible an assay for activity of mutant initiator tRNAs in vivo. We have identified the critical elements in the tRNA necessary for specifying all of its distinctive properties including the role of the unique features common to all eubacterial initiator tRNAs. In identity swap experiments, we have shown that introduction of these unique features into E. coli elongator methionine tRNA converts it into a fully active initiator tRNA in vivo.
In the process of this work, we have generated mutant initiator tRNAs which are blocked at each of the steps in the initiation pathway. Using these mutants, we have identified intragenic and extragenic suppressors which can rescue the initiation defects of some of the mutant tRNAs and have identified the important role of the amino acid methionine attached to the tRNA in recognition of the tRNA by the formylating enzyme and by the initiation factor IF2. We are now interested in structural analysis of the initiator tRNAs in a complex with various proteins such as the initiation factor IF2.
Other studies underway include an analysis of the requirements in initiator tRNA and in initiation factors for “de novo initiation”, which involves the assembly of mRNA, ribosome and initiator tRNA, versus “reinitiation”, in which the ribosome that has finished translation of an open reading frame and is bound to the mRNA, continues to translate the next open reading frame. We have identified some of the requirements in initiator tRNA and initiation factors for reinitiation.
Initiation of Protein Synthesis in Eukaryotes: The formation of a ternary complex between the initiator Met-tRNA, GTP and the translation initiation factor eIF2 is a crucial step in initiation of protein synthesis in eukaryotes. Our studies have identified the important role of a base pair conserved in eukaryotic initiator tRNAs in binding to eIF2. Other experiments have identified structural elements in mammalian initiator tRNA which block its participation in the elongation step in mammalian cells. We have also shown that protein synthesis can be initiated in mammalian cells with codons such as GUC and amino acids such as valine. Results of such studies provide opportunities for isolating suppressor mutations in yeast genes encoding components of the initiation machinery.
Other work along these lines involves a study of the very unusual phenomenon of “cap independent” initiation of protein synthesis without the involvement of the canonical initiator tRNA. This type of initiation is mediated by internal ribosome entry sequences present in some insect dicistronic viral RNAs. Using mutant reporters carrying UAG as the initiation codon and an amber suppressor tRNA, we have provided direct evidence that such initiation occurs from the ribosomal A site in mammalian cells in tissue culture.
Protein Synthesis in Archaea: We are also studying protein synthesis in archaea. The archaeal protein synthesis machinery is a mosaic with some features found in eubacteria and others found in eukaryotes. Mutant initiator tRNAs and protein coding genes carrying mutations in the initiation codon are being used to study the requirements in an archaeal initiator tRNA for its function. We have shown that, in the presence of a mutant initiator tRNA that can read the GUC codon, GUC can be used as an initiation codon in halobacteria.
We are also interested in understanding the mechanism by which an archaeal isoleucine tRNA reads the rare isoleucine codon AUA without also reading AUG, the codon for methionine. In eubacterial isoleucine tRNA, the modified base lysidine derived from cytidine, is used to exclusively base pair with A. In eukaryotic isoleucine tRNAs, modified bases pseudouridine and inosine – each found in one of the two isoleucine tRNAs – can base pair with A without base pairing with G. We have recently found that the archaeal isoleucine tRNA, which reads AUA, also has a modified base derived from cytidine, but it is not lysidine, pseudouridine, or inosine. Current work is aimed at identification of the modified base and enzymes involved in its biosynthesis.
Suppressor tRNAs in Mammalian Cells: With the objective of isolating animal viruses with nonsense mutations in their genes, we previously established mammalian cell lines carrying an inducible amber suppressor tRNA gene (with Phillip Sharp). We are developing more generally applicable systems for inducible expression of tRNA genes and/or tRNA function. One approach is to introduce genes for suppressor tRNAs which are not substrates for mammalian aminoacyl-tRNA synthetases, and express a cognate aminoacyl-tRNA synthetase gene in an inducible manner. We have shown that amber, ochre and opal suppression in mammalian cells can be made dependent upon expression of the E. coli glutaminyl-tRNA synthetase (GlnRS) gene. We have demonstrated tetracycline-regulated expression of GlnRS and, thereby, tetracycline-regulated suppression of amber codons in mammalian cells. The next step is to isolate mammalian cell lines carrying such tRNAs and aminoacyl-tRNA synthetase genes.
Site-specific Incorporation of Unnatural Amino Acids into Proteins in vivo: We are working on developing methods for the incorporation of unnatural amino acids carrying fluorescent, photoaffinity or spectroscopic probes and heavy atoms, chemically reactive amino acids and phosphoamino acid analogues into proteins in vivo in eubacteria and in eukaryotic cells. Such proteins can be used for a variety of studies including the intracellular location of proteins, protein-protein interactions, signal transduction, analysis of structure, assembly and function of proteins and use of proteins as biosensors. A first step is the identification of:
- suppressor tRNAs that cannot be aminoacylated by any of the endogenous aminoacyl-tRNA synthetases, and
- aminoacyl-tRNA synthetases, which aminoacylate the suppressor tRNA but no other tRNA in the cell.
We have identified such orthogonal synthetase-tRNA pairs for use in E. coli, yeast and mammalian cells. We are now working on isolation of mutants in the aminoacyl-tRNA synthetase, which utilize the desired amino acid analogue instead of the normal amino acid. In collaboration with the group of Thomas Sakmar at Rockefeller University, we have recently described the incorporation of two chemically reactive keto amino acids at various sites of two G protein-coupled receptors, rhodopsin and CCR5.
We have also described a potentially more generally applicable system involving import into cells of suppressor tRNAs aminoacylated with the unnatural amino acid. The only requirement for this approach is that the suppressor tRNA that is imported should not be aminoacylated in the cells. We have identified such suppressor tRNAs and have imported amber (UAG) and ochre (UAA) suppressor tRNAs into mammalian cells. We have further shown that amber and ochre codons present in a reporter mRNA can be concomitantly suppressed by these tRNAs. These results open up the possibility of incorporating two different unnatural amino acids into the same protein in mammalian cells.
Bhattacharya*, A., Köhrer*, C., Mandal, D., and RajBhandary, U.L. Nonsense suppression in archaea. Proc. Natl. Acad. Sci. USA 112:6015-6020 (2015; *authors contributed equally).
Sinha, A., Köhrer, C., Weber, M.H., Masuda, I., Mootha, V.K., Hou, Y.M., and RajBhandary, U.L. Biochemical characterization of pathogenic mutations in human mitochondrial methionyl-tRNA formyltransferase. J. Biol. Chem. 289:32729-32741 (2014).
Vercruysse, M., Köhrer, C., Davies, B.W., Arnold, M.F., Mekalanos, J.J., and RajBhandary, U.L., and Walker, G.C. The highly conserved bacterial RNase YbeY is essential in Vibrio cholerae, playing a critical role in virulence, stress regulation, and RNA processing. PloS Pathog.10:1-20 (2014).
Mandal*, D., Köhrer*, C., Su, D., Babu, I.R., Chan, C.T., Liu, Y., Söll, D., Blum, P., Kuwahara, M., Dedon, P.C., and RajBhandary, U.L. Identification and codon reading properties of 5-cyanomethyl uridine, a new modified nucleoside found in the anticodon wobble position of mutant haloarchaeal isoleucine tRNAs. RNA 20:177-188 (2014; *authors contributed equally).
Köhrer, C., Mandal D., Gaston, K.W., Grosjean, H., Limbach, P.A., and RajBhandary, U.L. Life without tRNAIle-lysidine synthetase: translation of the isoleucine codon AUA in Bacillus subtilis lacking the canonical tRNA2Ile. Nucleic Acids Res. 42:1905-1915 2014).
Voorhees, R.M., Mandal, D., Neubauer, C., Köhrer, C., RajBhandary, U.L., and Ramakrishnan, V. The structural basis for specific decoding of AUA by isoleucine tRNA on the ribosome. Nat. Struct. Mol. Biol. 20:641-643 (2013).
Jacob, A.L., Köhrer, C., Davies, B.W., RajBhandary, U.L., and Walker, G.C. Conserved bacterial RNase YbeY plays key roles in 70S ribosome quality control and 16S rRNA maturation. Mol. Cell 49:427-38 (2013).
Tucker*, E.J., Hershman*, S.G., Köhrer*, C., Belcher-Timme, C.A., Patel, J., Goldberger, O.A., Christodoulou, J., Silberstein, J.M., McKenzie, M., Ryan, M.T., Compton, A.G., Jaffe, J.D., Carr, S.A., Calvo, S.E., RajBhandary, U.L., Thorburn, D.R., and Mootha, V.K. ). Mutations in MTFMT underlie a human disorder of formylation causing impaired mitochondrial translation. Cell Metab. 14:428-434. (2011; *authors contributed equally).
Mandal*, D., Köhrer*, C., Su, D., Russell, S.P., Krivos, K., Castleberry, C.M., Blum, P., Limbach, P.A., Söll D., and RajBhandary, U.L. Agmatidine, a modified cytidine in the anticodon of archaeal tRNAIle, base pairs with adenosine but not with guanosine. U.L. Proc. Natl. Acad. Sci. USA 107:2872-2877. (2010; *authors contributed equally).
Kamoshita, N., Nomoto, A., and RajBhandary, U.L. Translation initiation from the ribosomal A site or the P site, dependent on the conformation of RNA pseudoknot I in dicistrovirus RNAs. Mol. Cell 35:181-190. (2009).
“Protein Engineering” edited by Köhrer, C. and RajBhandary, U.L. Nucleic Acids and Molecular Biology, Vol. 22:347 Springer (2009).
Yoo, J-H. and U. L. RajBhandary. Requirements for translation re-Initiation in Escherichia coli: Roles of initiator tRNA and initiation factors IF2 and IF3. Mol. Microbiol. 67:1012-1026 (2008).
Köhrer, C., Srinivasan, G., Mandal, D., Mallick, B., Ghosh, Z., Chakrabarti, J., and RajBhandary, U.L. Identification and characterization of a tRNA decoding the rare codon AUA in Haloarcula marismortui. RNA 14:117-126 (2008).
Ye*, S., Köhrer*, C., Huber, T., Kazmi, M., Sachdev, P., Yan, E.C.Y., Bhagar, A., RajBhandary, U.L., and Sakmar T.P. Site-Specific incorporation of keto amino acids into functional G protein-coupled receptors using unnatural amino acid mutagenesis. J. Biol. Chem. 283:1525-1533 (2008; *authors contributed equally).