Understanding ribosome biogenesis

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UNDERSTANDING RIBOSOME BIOGENESIS The overall objective of my research program is to understand how bacteria grow and survive. In the proposed research we will focus on the most basic of questions, to understand how the ribosome is assembled. The Escherichia coli ribosome is the product of assembly of a 16S ribosomal RNA and 21 ribosomal proteins for the 30S subunit, as well as a 5S rRNA, a 23S rRNA, and 34 proteins for the 50S subunit. Vintage studies by Nomura’s laboratory more than forty years ago revealed that functional ribosomal subunits could be assembled in a test tube with its component ribosomal RNAs and proteins. These and more contemporary studies have significantly advanced our understanding of the forces shaping subunit assembly. However, where these experiments have been performed under non-physiological conditions and times, it is noteworthy that E. coli assembles a ribosome extremely rapidly – in about two minutes – and it does so with more than 50 non-ribosomal protein factors. It is difficult to study ribosome assembly in vivo because assembly intermediates do not accumulate in amounts that permit isolation. Nevertheless, factors assisting ribosomal assembly represent extraordinary probes of ribosome maturation. Mutations directed at these factors can disable ribosome biogenesis and make it possible to isolate and characterize the products of perturbation. This approach is facilitating challenging studies of subtle aspects of folding and assembly that are the focus of this proposal. Most interestingly, an emerging group of biogenesis proteins, so-called ‘enigmatic factors’, are GTPases and RNA-binding proteins that appear to have chaperone or checkpoint roles in the late steps of the assembly process. Much of our work to date has focused on the YjeQ protein and has revealed a role in late steps of 30S subunit assembly. Evidence is mounting that several biogenesis factors constitute a functional network and prevent premature entry of late assembly intermediates into the translating pool of 70S ribosomes. For the 30S subunit the focus is on a cast of enigmatic factors that currently include BipA, Era, RbfA, RimM, RimP and YjeQ. A limited genetic interaction study in my laboratory revealed that overexpression of Era, a GTPase with a putative role in 30S assembly, was a suppressor of the slow growth and ribosome defects of the yjeQ deletion strain. Similarly, loss of the enigmatic 30S assembly factor RimM led to enhancement of these phenotypes. In the research proposed herein, we will expand our efforts to chart a genome-wide map of the functional interactions of these enigmatic factors in addition to exploring the implications with further studies of the assembly of the 30S ribosomal subunit. Our short-term goals are as follows: Objective 1. Probe the functional network of enigmatic factors with roles in 30S biogenesis. Objective 2. Test hypotheses posed by the functional network of enigmatic factors. Objective 3. Determine the nature of subunits that accumulate on depletion of biogenesis factors Where ribosome biogenesis is increasingly thought to proceed through multiple, parallel pathways guided by dispensable and redundant assembly factors, the proposed systems approach will be key to making impactful progress. My group’s unique capabilities in high throughput biology and conventional biochemistry will permit us to describe the network with systems approaches and to test the implications with reductionist experiments. I believe this argues strongly for the potential impact of the proposed work and its prospects to equip trainees with outstanding and varied skills in discovery research.