Poster abstracts

Poster number 43 submitted by Ruisheng Jiang

Function of the N-terminal domain of Pyrrolysyl-tRNA synthetase

Ruisheng Jiang (Microbiology, OSBP)

Abstract:
Pyrrolysine (Pyl) is an atypical amino acid encoded by the UAG codon in methylamine methyltransferase genes of Methanosarcina barkeri. Pyrrolysyl-tRNA synthetase (PylRS) is the specific enzyme that can attach Pyl to its cognate tRNA, the amber suppressor tRNApyl. Besides the archaeal family Methanosarcinaceae, genes that encode PylRS are also found in Desulfitobacterium hafniense, a Gram-positive bacterium. The pylSc gene produces a D. hafniense PylRS (PylSc) that is about 100 amino acids shorter than the Methanosarcinaceae PylRS proteins. Although D. hafniense PylSc has the in vitro aminoacylation activity, it has very limited in vivo activity as a PylRS relative to the Archaeal enzymes. Downstream of the D. hafniense pyl gene cluster is the pylSn gene encoding a 110 residue protein. The PylSn protein has over 50% sequence similarity to the N-terminal portion of Methanosarcinaceae PylRS, which is critical for in vivo activity of PylRS. PylSc has catalytic activity but binds tRNApyl with poorer affinity than does archaeal PylRS. PylSn has tRNApyl binding activity as assayed by gel retardation, but does not bind D. hafniense tRNAAla, tRNALys,, or tRNAPhe transcripts using the same assay. In contrast, lower affinity binding by PylSn of these other tRNAs could be detected by filter binding. PylSn also showed as high of tRNApyl binding affinity as an archaeal PylRS. An N-terminally truncated M. barkeri PylRS had decreased tRNApyl affinity but retained partial catalytic activity. PylRS from D. hafniense is thus apparently composed of two different gene products, with PylSc comprising the catalytic portion with low tRNA binding affinity, and PylSn containing a higher affinity tRNA binding domain.

Keywords: Pyrrolysine, tRNA, synthetase