Laboratory of Microbial Biochemistry

Skidmore College

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Research Projects

Attaching an amino acid to its cognate tranfer RNA (tRNA) establishes the genetic code. Errors in the process decrease the fidelity of translation. We are interested in how the amino acid asparagine (Asn) is ligated to tRNAAsn. The amino acid can be directly attached to the tRNA using an asparaginyl-tRNA synthetase (AsnRS). However, most prokaryotes lack AsnRS and instead use a two-step indirect pathway to form Asn-tRNAAsn. These organisms take advantage of an aspartyl-tRNA synthetase (AspRS) with relaxed tRNA specificity, recognizing both the tRNA for aspartate (Asp) and Asn. In the indirect pathway this non-discriminating AspRS (ND-AspRS) attaches Asp to tRNAAsn. The Asp is then amidated by the amidotransferase GatCAB to Asn to form Asn-tRNAAsn used in protein synthesis. In organisms lacking a glutaminyl-tRNA synthetase (GlnRS), a similar pathways exist to form Gln-tRNAGln. A non-discriminating glutamyl-tRNA synthetase (ND-GluRS) attaches glutamate (Glu) to tRNAGln and an amidotransferase converts the Glu to glutamine (Gln) on the tRNA. In bacteria the amidotransferase for Gln-tRNAGln formation is GatCAB and in archaea it is GatDE. We are interested in how these pathways fit into bacterial life cycles. The work will help us understand how bacteria adapt to stress, how fidelity of protein synthesis is maintained under such conditions, and how decoding pathways evolved providing insight into the evolution of the genetic code. In addition, the work will lay the foundation for the development of novel antibiotics and new tools for synthetic biology. As proof of principle for the latter, we are using our knowledge of these pathways to expand the genetic code.

RNA-Dependent Asn Biosynthesis in S. aureus

We have demonstrated the human pathogen Staphylococcus aureus uses both the direct and indirect routes for Asn-tRNAAsn formation. We are interested in understanding why this is the case.

 

Given S. aureus lacks the Asn synthetases (AsnA and AsnB) to amidate Asp to Asn, we predict S. aureus only forms Asn on tRNAAsn via the two-step pathway. We are using molecular genetic techniques and gene expression studies to test this hypothesis and explore the role of AsnRS in S. aureus given the presence of the indirect route. The work is supported by funding from the National Science Foundation.

RNA-Dependent Asn Biosynthesis in Bacilli

Bacilli in their genomes encode an AsnRS, AsnB, and GatCAB but not GlnRS. Given they have AsnB to make Asn that AsnRS can then ligate to tRNAAsn but no GlnRS, the predicted role of GatCAB in Bacilli is for Gln-tRNAGln formation. However, we have evidence that the AspRS enzymes in certain Bacilli are non-discriminating and can work in concert with GatCAB to synthesize Asn in a tRNA-dependent manner. The work is supported by funding from the National Science Foundation.

   

Why these bacteria have different routes for Asn and Asn-tRNAAsn biosynthesis is unclear. We are using biochemical and molecular genetic techniques to better understand these pathways in Bacilli.

Evolution of Bacterial Aspartyl-tRNA Synthetase Specificity

How many other bacteria use both the direct and indirect pathways for Asn-tRNAAsn formation is unclear as we can not predict based on sequence whether an AspRS is discriminating or not. The image below is of the E. coli discriminating AspRS. How it evolved to distinguish tRNAAsp from tRNAAsn is unknown. Understanding that structure-function relationship will allow us to better predict which bacteria use both pathways for Asn-tRNAAsn synthesis.

   

To understand how discriminating AspRS evolved we are using a combination of structural biology, biochemistry, and molecular phylogenetics.

Expansion of the Genetic Code

When it comes to studying protein function and reengineering of organisms, it would be advantageous to expand the genetic code beyond the twenty-two amino acids used by life currently in protein synthesis. Most of the work in the area has centered around altering the amino acid specificity of aminoacyl-tRNA synthetases. We are interested in applying our understanding of the indirect pathways to synthesize novel amino acids on suppressor tRNAs.

   

In particular we are interested in expanding the genetic code with L-oxo-proline by reengineering E. coli using our understanding of the indirect two-step pathways. The work is supported by funding from the Research Corporation.