Phenotypic effects of paralogous ribosomal proteins bL31A and bL31B in E. coli.

Ribosomes are essential macromolecular complexes conducting protein biosynthesis in all domains of life. Cells can have heterogeneous ribosomes, i.e. ribosomes with various ribosomal RNA and ribosomal protein (r-protein) composition. However, the functional importance of heterogeneous ribosomes has remained elusive. One of the possible sources for ribosome heterogeneity is provided by paralogous r-proteins. In E. coli, ribosomal protein bL31 has two paralogs: bL31A encoded by rpmE and bL31B encoded by ykgM. This study investigates phenotypic effects of these ribosomal protein paralogs using bacterial strains expressing only bL31A or bL31B. We show that bL31A confers higher fitness to E. coli under lower temperatures. In addition, bL31A and bL31B have different effects on translation reading frame maintenance and apparent translation processivity in vivo as demonstrated by dual luciferase assay. In general, this study demonstrates that ribosomal protein paralog composition (bL31A versus bL31B) can affect cell growth and translation outcome.

Bacterial ribosome heterogeneity: Changes in ribosomal protein composition during transition into stationary growth phase.

Ribosomes consist of many small proteins and few large RNA molecules. Both components are necessary for ribosome functioning during translation. According to widely accepted view, bacterial ribosomes contain always the same complement of ribosomal proteins. Comparative bacterial genomics data indicates that several ribosomal proteins are encoded by multiple paralogous genes suggesting structural heterogeneity of ribosomes. In E. coli, two r-proteins bL31 and bL36 are encoded by two genes: rpmE and ykgM encode bL31 protein paralogs bL31A and bL31B, and rpmJ and ykgO encode bL36 protein paralogs bL36A and bL36B respectively. We have found several similarities and differences between ribosomes of exponential and stationary growth phases by using quantitative mass spectrometry and X-ray crystallography. First, composition of ribosome associating proteins changes profoundly as cells transition from exponential to stationary growth phase. Ribosomal core proteins bL31A and bL36A are replaced by bL31B and bL36B, respectively. Second, our X-ray structure of the 70S ribosome demonstrates that bL31B and bL36B proteins have similar ribosome binding sites to their A counterparts. Third, ribosome subpopulations containing A or B paralogs existed simultaneously demonstrating that E. coli ribosomes are heterogeneous with respect to their paralogous ribosomal protein composition that changes via protein exchange.

The Intersubunit Bridge B1b of the Bacterial Ribosome Facilitates Initiation of Protein Synthesis and Maintenance of Translational Fidelity.

In bacteria, ribosomal subunits are connected via 12 intersubunit bridges involving RNA-RNA, RNA-protein, and protein-protein interactions. The only protein-protein bridge in the ribosome is ribosomal intersubunit bridge 1b (B1b), which is mainly formed by the bacterial protein L31 (bL31) and connects the head domain of 30S subunit and the central protuberance of the 50S subunit. It is known to be the most dynamic intersubunit bridge. Here, we have evaluated the role of bL31 and thereby the bridge B1b in the working cycle of the ribosome. First, bL31-deficient ribosomes are severely compromised in their ability to ensure translational fidelity particularly in reading frame maintenance in vivo. Second, in the absence of bL31, the rate of initiation is significantly reduced both in vivo and in vitro. Third, polysome profile and subunit reassociation assays demonstrate that bL31 is important for stabilizing subunit joining in vivo and in vitro. Together, our results demonstrate that bL31 is important for determining translational fidelity and stabilizing subunit association. We conclude that the only protein-protein intersubunit bridge of the bacterial ribosome facilitates translation initiation and is essential for maintaining the reading frame of mRNA translation.