Overview

Our group is interested in how the ribosome works. The ribosome is a large (~2.5 MDa), two-subunit, RNA-based machine that translates the genetic code in all organisms. In recent years, numerous structures of the ribosome and various ribosomal complexes have been determined by X-ray crystallography and cryo-electron microscopy. Today, a primary challenge is to understand how the ribosome moves and how such dynamics govern the various steps of translation. Since the ribosome is the most common target of natural antibiotics, gaining insight on ribosome function may contribute substantially to the development of new antibiotics.

We use a combination of genetic, molecular, and biochemical methods to study protein synthesis in bacteria. Examples of questions under investigation in the laboratory include: (1) Which features of mRNA tune the rate of initiation? And how do the mechanisms of initiation compare among the different bacterial phyla? (2) How do rRNA dynamics contribute to the mechanism of decoding (aminoacyl-tRNA selection)? (3) What roles do nonessential ribosome-associated GTPases play in the cell?

Determining the roles of conserved translational GTPases in ribosome assembly

Translational GTPases (trGTPases) are fundamental proteins that couple GTP hydrolysis to molecular events on the ribosome. Many bacterial trGTPases, such as IF2, EF-G, EF-Tu, and RF3, play well known roles in the cell. The functions of LepA and BipA, however, have remain enigmatic. Recent studies provides compelling evidence that LepA and BipA function in biogenesis of the 30S and 50S subunit of the ribosome, respectively. Using a SILAC and mass spectrometry approach, we determined that in the absence of LepA, an immature 30S particle forms that lacks small subunit proteins S3, S10, S14, and S21. All these proteins bind late in the assembly process and facilitate folding of the 3′ domain of 16S rRNA (see figure). Processing of 16S rRNA is also delayed in the mutant strain as indicated by increased levels of precursor rRNA in the assembly intermediates. Previous work suggested BipA plays a role in 50S assembly, but details have yet to be elucidated. We are currently using our SILAC approach to understand the protein composition of immature assembly intermediates that accumulate in the absence of BipA. Together these results offer important implications for understanding ribosome assembly in bacteria. Particularly given LepA and BipA are translation GTPases that function in the context of the 70S ribosome, suggesting the final stages of assembly may occur in a 70S ribosome.

Translation initiation in the peculiar phylum, the Bacteroidota.

Typically in bacteria (i.e. E. coli), translation initiation uses an interaction between the 3′ end of the 16S rRNA, the Anti-Shine Dalgarno (ASD), and the 5′ UTR of the message, the Shine Dalgarno (SD). This base pairing between the 16S and the message helps to position the message so that the start codon is in the P-site of the 30S subunit. In the phylum Bacteroidota though SD sequences are missing yet the ASD of the 16S is conserved. We have shown the determinants that a Bateroidota representative, Flavobacterium johnsoniae (Fjo), use to fine tune translation initiation of Fjo messages. We have also showed that Fjo 30S subunits are “blind” to SD sequences due to the 3′ end of the 16S being sequestered in a pocket formed by ribosomal proteins S6, S18 and S21. However, the gene rpsU, which encodes S21, contains a strong SD. The story as to why Fjo retains the SD sequence in the rpsU gene has been published and can be found here (1).