Enzymatic basis for cytidine to uridine editing on archaeal tRNAs

• Applicant: Dr. Ilka Heinemann
• Subject Area: Metabolism, Biochemistry and Genetics of Microorganisms ; Parasitology and Biology of Tropical Infectious Disease Pathogens
• Term: from 2008 to 2011
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 86156855

• Final Report Year: 2012

Final Report Abstract

In summary, the following aims have been reached during my appointment at Yale. 1. I provided the first identification and characterization of homologs of tRNAHis guanylyltransferase (Thg1) from both bacteria and archaea. Prior to my work on this enzyme, Thg1 had only been identified in eukaryotes. Prokaryotic variants differ from the eukaryotic variants in their substrate specificity and might be useful in further protein engineering attempts to create a true reverse polymerase. 2. Characterization of Pyrrolysine as a regular amino acid in Thg1 and analysis of the Pyl-protome in both archaea and bacteria. This work broadened the view of Pyrrolysine as a regular amino acid and corrected the view of Pyl incorporation from site directed genetic mRNA recoding to undirected Stop codon suppression. 3. Creation of a genetically modified Escherichia coli strain to enable efficient UAG recoding to phosphoserine. By eliminating the detrimental competition between suppression and chain termination via deletion of release factor 1, we were able to create a highly useful tool, applicable in the production of proteins encoding unnatural amino acids such as phophoserine.

Publications

• (2009). The appearance of pyrrolysine in tRNAHis guanylyltransferase by neutral evolution. Proc Natl Acad Sci USA. 106(50):21103-8
  Heinemann, I.U., O'Donoghue, P., Madinger, C., Benner, J., Randau, L., Noren, C.J., and Söll, D.
  
• (2010). 3'-5' tRNAHis guanylyltransferase in bacteria. FEBS Lett. 584(16):3567-72
  Heinemann, I.U., Randau, L., Tomko Jr, J.R., and Söll, D.
  
• (2010). Transfer RNA processing in archaea: unusual pathways and enzymes. FEBS Lett. 584(2):303-9
  Heinemann, I.U., Söll, D. and Randau, L.
  
• (2011). Alanyl-phosphatidylglycerol synthase: mechanism of substrate recognition during tRNA-dependent lipid modification in Pseudomonas aeruginosa. Mol Microbiol. 80(4):935-50
  Hebecker, S., Arendt, W., Heinemann, I.U., Tiefenau, J.H., Nimtz, M., Rohde, M., Söll, D. and Moser, J.
  
• (2012). Enhanced phosphoserine insertion during Escherichia coli protein synthesis via partial UAG codon reassignment and release factor 1 deletion. FEBS Lett. [Epub ahead of print]
  Heinemann, I.U., Rovner, A.J., Aerni H.R., Rogulina, S., Cheng, L., Olds, W., Fischer, J.T., Söll, D., Isaacs, F.J. and Rinehart, J.
  (See online at https://doi.org/10.1016/j.febslet.2012.08.031)
• (2012). Near-cognate suppression of amber, opal and quadruplet codons competes with aminoacyl-tRNAPyl for genetic code expansion. FEBS Lett. [Epub ahead of print]
  O’Donoghue, P., Prat, L., Heinemann, I.U., Ling, J., Odoi, K., Huang, Y., Liu, W.R. and Söll, D.
  (See online at https://doi.org/10.1016/j.febslet.2012.09.033)
• (2012). tRNAHis guanylyltransferase establishes tRNAHis identity. Nucleic Acids Res. 40: 333-344
  Heinemann, I.U., Nakamura, A., O’Donoghue, P., Eiler, D. and Söll, D.