Key ribosome assembly work [additional here]

1. KsgA facilitates ribosomal small subunit maturation  by proofreading a key structural lesion. 

Sun JY*, Kinman LK*, Ortega J, Davis JH. Under review [pdf]

This tour-de-force applies the full suite of cryo-EM analysis methods we’ve developed to understand how a methyltransferase (KsgA) facilitates bacterial ribosome biogenesis. We find that KsgA performs a novel “proofreading” function, promoting the selective disassembly of particles that are largely mature, but bear subtle structural lesions making them inactive. This surprising result harkens back to classic models of kinetic proofreading (Hopfield, 1974) first hypothesized for polymerases and tRNA synthetases, and newly extends this mechanism to bacterial ribosome assembly.

Using the approaches we present here, we can quantitatively compare hundreds of maps sampled from a single dataset, and can furthermore compare the resulting structural landscapes across datasets. We expect this general workflow to be widely adopted to analyze all sorts of molecular machines imaged by single particle cryo-EM.

Modular Assembly of the Bacterial Large Ribosomal Subunit.

Davis JH, Tan YZ, Carragher B, Potter CS, Lyumkis D, Williamson JR. Cell 2016. [pubmed] [pdf]

This work uses key MS and EM tools we’ve developed, and led to significant advances in understanding ribosome biogenesis. For example, we found that (i) the order of r-protein binding is flexible and can be ‘re-routed’, (ii) limitation of a single r-protein has long-range structural effects, (iii) ribosome assembly occurs through the coordinated and cooperative folding of large structural ‘blocks’, (iv) subunit assembly is initiated on the solvent face of the particle and can proceed along three parallel pathways, and (v) RNA misfolding events are common.

These studies seeded our interest in reconstructing highly heterogeneous structural ensembles, and this dataset serves as a “benchmarking” dataset for EM software tools developed all over the world.

Functional domains of the 50S subunit mature late in the assembly process

Jomaa A*, Jain N*, Davis JH*, Williamson JR, Britton RA, Ortega J. Nucleic Acids Research 2014. [pubmed] [pdf]

Here, we describe a mass spectrometry-based method an analysis framework to distinguish between dead-end and viable intermediates. This method measures in vivo assembly kinetics on a protein-by-protein basis in wild-type or assembly-factor depleted cells. Inspection of these measured assembly times allows proteins to be placed in a rough binding order, and, in some instances, can identify the rate-limiting assembly step. Moreover, by determining the size of the pools of assembly precursors, we can quantitatively assess what fraction of the accumulating ribosomal particles are competent for maturation.

Discovery of a small molecule that inhibits bacterial ribosome biogenesis

Stokes JM, Davis JH, Mangat CS, Williamson JR, Brown ED. eLife 2014. [pubmed] [pdf]

Due to widespread antibiotic resistance, there is a clear need to identify essential, bacteria-specific processes that can be targeted by next-generation antibiotics. To identify agents inhibiting ribosome biogenesis, and to validate ribosome assembly as a drug target, we designed a small-molecule screen to enrich for ribosome assembly inhibitors and identified the small-molecule Lamotrigine (Lam) potentially inhibited growth of E. coli. Using qMS and biochemistry, we found that Lam directly inhibited a novel assembly-related role for the translation GTPase IF-2. Critically, we used PL-qMS to measure protein-synthesis rates in various drug-treated cells directly, which demonstrated that Lam inhibited assembly and not protein synthesis, thereby providing the first validation of ribosome biogenesis as an antibiotic target.

Key Proteolysis work

Single-molecule denaturation and degradation of proteins by the AAA+ ClpXP protease.

Shin Y*, Davis JH*, Brau RR*, Martin A*, Kenniston JA, Baker TA, Sauer RT, Lang MJ. PNAS 2009. [pubmed] [pdf]

Here we developed a simple-molecule TIRF (smTIRF) assay to monitor ClpXP-mediated mechanical unfolding of protein substrates, their translocation, and their eventual degradation. This platform has since been expanded using optical tweezers to measure force exerted on substrates by ClpX, translocation step sizes, and other key biochemical and biophysical parameters of this molecular machines. In collaboration with the Sauer lab, we are now using cryoEM to begin to visualize structures of functional states predicted by these assays.

1) Small-molecule control of protein degradation using split adaptors.

Davis JH, Baker TA, Sauer RT. ACS Chemical Biology 2011. [pubmed] [pdf]

2) Engineering synthetic adapters and substrates for controlled ClpXP degradation.

Davis JH, Baker TA, Sauer RT. Journal of Biological Chemistry 2009. [pubmed] [pdf]

The controlled protein degradation systems we developed allows users to genetically tag a protein of interest with a short peptide degron. Addition of a small molecule then activates a selective protease, leading to rapid (~minutes) removal of the tagged protein from the cell. This approach, and variants of it has been widely adopted both in academia and in industry.

Additional methodological work

1) Design, construction and characterization of a set of insulated bacterial promoters.

Davis JH, Rubin AJ, Sauer RT. Nucleic Acids Research 2011. [pubmed] [pdf]

2) Measuring the activity of BioBrick promoters using an in vivo reference standard.

Kelly JR, Rubin AJ, Davis JH, Ajo-Franklin CM, Cumbers J, Czar MJ, de More K, Glieberman AL, Monie DD, Endy D. Journal of Biological Engineering 2009. [pubmed] [pdf]

Here, design and characterize a series of synthetic constitutive promoters for use in E. coli. These promoters were explicitly designed to be “insulated” from their surrounding sequence context, allowing for predicable levels of transcription independent of where they were inserted in the genome or in a plasmid. This library has been widely used in metabolic engineering, synthetic biology, and basic and translational science.

Additional ribosome assembly work

Structure and dynamics of bacterial ribosome biogenesis

Davis JH, Williamson JR. Philosophical Transactions of the Royal Society 2017. [pubmed] [pdf]

Here, we review our recent progress applying quantitative mass spectrometry and single particle cryo-electron microscopy to understand how bacterial ribosomes are assembled rapidly and efficiently. Additionally, we describe a quantitative framework to describe how perturbations to the biogenesis process may impact the accumulation of assembly intermediates and some of the potential pitfalls in interpreting these experiments.

1) Role of Era in assembly and homeostasis of the ribosomal small subunit

Razi A, Davis JH, Hao Y, Jahagirdar D, Thurlow B, Basu K, Jain N, Gomez-Blanco J, Britton RA, Vargas J, Guarne A, Woodson SA, Williamson JR, Ortega J. Nucleic Acids Research 2019. [pubmed] [pdf]

2) YphC and YsxC GTPases assist the maturation of the central protuberance, GTPase associated region and functional core of the 50S ribosomal subunit

Ni X, Davis JH, Jain N, Razi A, Benlekbir S, McArthur AG, Rubinstein JL, Britton RA, Williamson JR, Ortega J. Nucleic Acids Research 2016. [pubmed] [pdf]

3) Binding properties of YjeQ (RsgA), RbfA, RimM and Era to assembly intermediates of the 30S subunit

Thurlow B, Davis JH, Leong V, Moraes TF, Williamson JR, Ortega J. Nucleic Acids Research 2016. [pubmed] [pdf]

4) Functional interaction between ribosomal protein L6 and RbgA during ribosome assembly

Gulati M, Jain N, Davis JH, Williamson JR, Britton RA. PLoS Genetics 2014. [pubmed] [pdf]

These papers describe our collaborative efforts to understand how chaperones aid the ribosome assembly process - making it rapid, efficient, and error-free. In E. coli and B. subtilis, we use genetic perturbations (knockouts, depletions, suppressors, and over-expression strains) to modulate the biogenesis process, biochemistry and isolate and characterize the resulting assembly intermediates, quantitative mass spectrometry to determine the composition of these intermediates, and cryo-EM to determine the ensemble of structures these intermediates adopt. By comparing intermediates produced by various perturbations, we formulate testable hypotheses for how the assembly chaperones act.