Translation
RIBOSOME
The ribosome translates the genetic code of a mRNA into a polypeptide chain, a fundamental cell function. The functional ribosome consists of two subunits, a 50S and a 30S. In the Mtb ribosome, the 50S subunit is composed of the 23S rRNA, 5S rRNA, and 35 proteins while the 30S subunit contains a 16S rRNA and 22 proteins. The 30S contains a messenger RNA decoding center and the 50S contains a peptidyl transferase center. The cryo-electron microscopy images of Mtb ribosome reveal species-specific structural features, such as extended or inserted rRNA helices and several significantly longer rProteins. The most striking of these is a unique 100-nt long expansion segment of the Mtb 23S rRNA, named H54a or the ‘handle’, is flexible and change its conformation upon joining of the large and small ribosomal subunits.
Protein synthesis can be divided into four main steps: initiation, elongation, termination and recycling with appropriate factors required to accomplish each step. The essential nature of the ribosome in protein synthesis makes it an important but challenging target for antibiotics with a vast array of natural and synthetic inhibitors. Additionally, the translation factors and aminoacyl tRNA synthetase (aaRSs), which are indispensable in translation, are also vital targets for antibiotics.
We have developed a translation assay that uses the Mtb ribosome to translate nanoluciferase mRNA in an Mtb-derived cell-free extract. The assay has been applied to screen potential potent inhibitors against Mtb translation system. Aside from wild type ribosome, we also induced methylated ribosome purified from Mtb treated with erythromycin.
Erythromycin induces the methyltransferase gene Erm (37) which methylate nucleotides A2296 (corresponded to A2058 in E. coli) in ribosome 23S rRNA. The methylated Mtb ribosome confers significant resistance to macrolide, lincosamide, and streptogramin B (MLSB), but no resistance to the latest macrolide derivatives.
Mtb has incredibly sophisticated approaches to fine-tune translation. It has the ability to enter a non-replicating state in which protein synthesis is presumed to be greatly diminished to conserve cellular resources. A ribosomal silencing factor during starvation or the stationary phase (RsfS) has been shown to slow down or block translation entirely.
The crystal structure of Mtb RsfS, together with the cryo-EM structure of the large subunit 50S of the Mtb ribosome solved in collaboration with Dr. Junjie Zhang, reveals it binds to the 50S at protein L14 and blocks the association of the small 30S subunit. It appears to play an important role in the maintenance of sustainable energy levels during nutritional shortages.
