Biology subjects

Rafferty, J. B.

Publications and source records attributed to Rafferty, J. B..

3 recordsLinked to original sources

bioRxiv2024

Expanding the RNA polymerase biocatalyst solution space for mRNA manufacture

All mRNA products are currently manufactured in in vitro transcription (IVT) reactions that utilize single-subunit RNA polymerase (RNAP) biocatalysts. Although it is known that discrete polymerases exhibit highly variable bioproduction phenotypes, including different relative processivity rates and impurity generation profiles, only a handful of enzymes are generally available for mRNA biosynthesis. This limited RNAP toolbox restricts strategies to design and troubleshoot new mRNA manufacturing processes, which is particularly undesirable given the continuing diversification of mRNA product lines towards larger and more complex molecules. Herein, we describe development of a high-throughput RNAP screening platform, comprising complementary in silico and in vitro testing modules, that enables functional characterisation of large enzyme libraries. Utilizing this system, we identified eight novel sequence-diverse RNAPs, with associated active cognate promoters, and subsequently validated their performance as recombinant enzymes in IVT-based mRNA production processes. By increasing the number of available characterized functional RNAPs by > 130% and providing a platform to rapidly identify further potentially useful enzymes, this work significantly expands the RNAP biocatalyst solution space for mRNA manufacture, thereby enhancing capability to achieve application and molecule-specific optimisation of product yield and quality.

bioengineering↗

bioRxiv2022

Conformational dynamics of cohesin/Scc2 loading complex are regulated by Smc3 acetylation and ATP binding

The ring-shaped cohesin complex is the key player in sister chromatid cohesion, DNA repair, and gene transcription. The loading of cohesin to chromosomes requires the loader Scc2 and is regulated by ATP. This process is also hindered by Smc3 acetylation. However, the molecular mechanism underlying this inhibition remains mysterious. Here we identify a novel configuration of Scc2 with pre-engaged cohesin and reveal dynamic conformations of the cohesin/Scc2 complex in the loading reaction. We demonstrate that Smc3 acetylation blocks the association of Scc2 with pre-engaged cohesin by impairing the interaction of Scc2 with Smc3s head. Lastly, we show that ATP binding induces the cohesin/Scc2 complex to clamp DNA by promoting the interaction between Scc2 and Smc3 coiled coil. Our results illuminate a dynamic reconfiguration of the cohesin/Scc2 complex during loading and indicate how Smc3 acetylation and ATP regulate this process.

biochemistry↗

bioRxiv2021

(p)ppGpp inhibits 70S ribosome formation in Staphylococcus aureus by impeding GTPase-ribosome interactions

During nutrient limitation, bacteria produce the alarmones (p)ppGpp as effectors of the stress signalling network termed the stringent response. Screening for (p)ppGpp-binding targets within Staphylococcus aureus identified four ribosome-associated GTPases (RA-GTPases), RsgA, RbgA, Era and HflX, each of which are cofactors in ribosome assembly, where they cycle between the ON (GTP-bound) and OFF (GDP-bound) states. Entry into the OFF-state from the ON-state occurs upon hydrolysis of GTP, with GTPase activity increasing substantially upon ribosome association. When bound to (p)ppGpp, GTPase activity is inhibited, reducing 70S ribosome assembly. Here, we sought to determine how (p)ppGpp impacts RA-GTPase-ribosome interactions by examining the affinity and kinetics of binding between RA-GTPases and ribosomes in various nucleotide-bound states. We show that RA-GTPases preferentially bind to 5'-diphosphate-containing nucleotides GDP and ppGpp over GTP, which is likely exploited as a regulatory mechanism within the cell. Binding to (p)ppGpp reduces stable association of RA-GTPases to ribosomal subunits compared to the GTP-bound state both in vitro and within bacterial cells by inducing the OFF-state conformation. We propose that in this conformation, the G2/switch I loop adopts a conformation incompatible with ribosome association. Altogether, we highlight (p)ppGpp-mediated inhibition of RA-GTPases as a major mechanism of stringent response-mediated growth control.

microbiology↗