Biology subjects

Boël, G.

Publications and source records attributed to Boël, G..

3 recordsLinked to original sources

bioRxiv2024

Base composition at the start of the coding sequence controls the balance between translation initiation and mRNA degradation in E. coli .

Protein synthesis efficiency is highly dependent on the mRNA coding sequence. Furthermore, there is extensive evidence of a correlation between mRNA stability and protein expression level, though the mechanistic determinants remain unclear. Using yellow fluorescent protein (YFP) as a reporter gene, we herein demonstrate that adenosine (A) abundance in the first six codons is a critical determinant for achieving high protein synthesis in E. coli. Increasing A and/or decreasing guanosine (G) content in this region with synonymous codons results in substantial increases in protein expression level both in vivo and in vitro that are correlated with steady-state mRNA concentration in vivo. The change in mRNA concentration is attributable to changes in the stability of the mRNA that are directly coupled to its translation efficiency. Increasing A content promotes mRNA incorporation into the functional 70S ribosomal initiation complex without altering its affinity for the 30S ribosomal subunit. These results support a model in which base composition in the first six codons modulates local mRNA folding energy and single-strandedness to control the balance between productive translation initiation versus degradation of mRNAs bound to the 30S ribosomal subunit. Based on these findings, we developed a short N-terminal coding sequence that optimizes translation initiation efficiency for protein production in E. coli.

microbiology↗

bioRxiv2023

Cryo-EM studies of the four E. coli paralogs establish ABCF proteins as master plumbers of the peptidyl-transferase center of the ribosome

The genomes of most mesophilic organisms encode multiple ATP-Binding Cassette F (ABCF) proteins. EttA, one of four E. coli paralogs, regulates synthesis of the first peptide bond on the ribosome dependent on ATP/ADP ratio, while Antibiotic Resistance factors (AREs), paralogs in other organisms, both regulate and directly mediate resistance to ribosome-targeted antibiotics. However, the physiological functions remain unclear for most paralogs, and the mechanism-of-action has yet to be rigorously established for any paralog. We herein present single particle cryogenic electron microscopy structures of ribosome complexes of all four E. coli ABCF paralogs (EttA, Uup, YbiT, and YheS), which, together with previously determined ARE structures, show that ABCFs control the binding geometry of the tRNA in the peptidyl-tRNA-binding (P) site on the ribosome. They modulate the position of its acceptor stem relative to the peptidyl transferase center (PTC) in a manner that can either promote (EttA and Uup) or disrupt (YbiT, YheS, and the AREs) proper catalytic geometry. The YbiT/70S reconstructions include a conformation with no density for ribosomal protein bL33, and structural analyses support the exchange of this sub-stoichiometric ribosomal protein being functionally related to conformational changes in YbiT controlled by sequence variations in the strongly non-canonical Signature Sequence in its first ABC domain. Our studies establish general structural/enzymological principles by which the ATPase activity of ABCF proteins controls translation elongation coupled to modulation of conformation and stereochemistry in the catalytic core of the ribosome.

biophysics↗

bioRxiv2023

Comparative genetic, biochemical, and biophysical analyses of the four E. coli ABCF paralogs support distinct functions related to mRNA translation

Multiple paralogous ABCF ATPases are encoded in most genomes, but the physiological functions remain unknown for most of them. We herein compare the four Escherichia coli K12 ABCFs - EttA, Uup, YbiT, and YheS - using assays previously employed to demonstrate EttA gates the first step of polypeptide elongation on the ribosome dependent on ATP/ADP ratio. A {Delta}uup knockout, like {Delta}ettA, exhibits strongly reduced fitness when growth is restarted from long-term stationary phase, but neither {Delta}ybiT nor {Delta}yheS exhibits this phenotype. All four proteins nonetheless functionally interact with ribosomes based on in vitro translation and single-molecule fluorescence resonance energy transfer experiments employing variants harboring glutamate-to-glutamine active-site mutations (EQ2) that trap them in the ATP-bound conformation. These variants all strongly stabilize the same global conformational state of a ribosomal elongation complex harboring deacylated tRNAVal in the P site. However, EQ2-Uup uniquely exchanges on/off the ribosome on a second timescale, while EQ2-YheS-bound ribosomes uniquely sample alternative global conformations. At sub-micromolar concentrations, EQ2-EttA and EQ2-YbiT fully inhibit in vitro translation of an mRNA encoding luciferase, while EQ2-Uup and EQ2-YheS only partially inhibit it at ~10-fold higher concentrations. Moreover, tripeptide synthesis reactions are not inhibited by EQ2-Uup or EQ2-YheS, while EQ2-YbiT inhibits synthesis of both peptide bonds and EQ2-EttA specifically traps ribosomes after synthesis of the first peptide bond. These results support the four E. coli ABCF paralogs all having different activities on translating ribosomes, and they suggest that there remains a substantial amount of functionally uncharacterized "dark matter" involved in mRNA translation.

biochemistry↗