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"Put simply, our interest is the ribosome and how it is regulated."

What is the ribosome?

In every organism, translation of the genetic code into functional proteins is performed on the ribosome, a macromolecular machine of more than 2.3 MDa. In bacteria, the 70S ribosome is composed of two subunits, a small 30S subunit and a large 50S subunit. Each subunit is composed of ribosomal RNA (rRNA) and ribosomal proteins (r-proteins). In E. coli for example, the 30S subunit contains a single 1542 nucleotide (nt) 16S rRNA and 21 small subunit r-proteins (S-proteins), whereas the 50S subunit contains two RNAs, a 115 nt 5S rRNA and 2904 nt 23S rRNA, together with 33 large subunit r-proteins (L-proteins).

How do ribosomes synthesize polypeptides?

In bacteria, protein synthesis itself occurs on the 70S ribosome, and can be divided into three distinct phases; initiation, elongation and termination/recycling. Each of these phases has a specific set of translation factors that regulate the process. The initiation phase of translation in bacteria involves three initiation factors termed IF1, IF2 and IF3, which mediate the placement of the initiator transfer RNA (tRNA) and start codon of the messenger RNA (mRNA) at the P site of the ribosome (See 1a). The ribosome is now ready to enter into the elongation cycle. The codon following the start codon is displayed at the A site and dictates which amino-acyl-tRNA (aa-tRNA) will now bind. Aa-tRNAs are delivered to the A site in the form of a ternary complex consisting of an elongation factor EF-Tu, GTP and the aa-tRNA (1b). After GTP hydrolysis EF-Tu·GDP is released from the ribosome and the aa-tRNA docks into the A site (resulting in 1c). Peptide bond formation involves the transfer of the peptidyl moiety of the P-tRNA to the amino-acyl moiety of the A-tRNA (2): Note that the whole polypeptide chain is added to the new amino acid rather than the addition of the new amino acid to the chain. Peptide bond formation occurs on the large subunit at the peptidyl-transferase (PTF) center. Following peptide bond formation, no significant change in the positions of the two tRNAs is observed, although the P site now contains an uncharged tRNA and the A site contains a peptidyl-tRNA (2).

Transfer of the A and P-tRNAs to the P and E sites is termed translocation and is mediated by a second elongation factor EF-G. Put simply, the role of the elongation factors is to accelerate the elongation cycle to achieve the rate of 50 msec per elongation cycle in vivo. Without elongation factors the rate is about four orders of magnitude slower due to the high energy barrier (120 kJ/mol) that separates the pre- and post-translocational states (2 and 3, respectively) in E. coli ribosomes. Translocation places the deacylated tRNA at the E site and peptidyl-tRNA at the P site, freeing the A site for the binding of the next aa-tRNA (3). Binding of the next A-tRNA releases the E-tRNA (compare 1b and 1c) and so the cycle repeats (i.e. back to 3) until a stop codon appears in the A site. At this point protein termination factors RF1 or RF2 release the completed polypeptide, are recycled by RF3, and then the ribosome is dissociated into the component subunits by the action of the ribosome recycling factor (RRF), EF-G and IF3, in preparation for the next round of translation.

Ribosomes are a target for many antibiotics

Because ~50% of an E. coli cell’s total energy turnover goes towards protein synthesis, the translational apparatus must be strictly regulated. The fundamental importance of translation within the cell makes the ribosome a major target in the cell for natural antibiotics. Indeed, a plethora of drugs have been identified that target each and every step within the translation cycle.

In order to address how specific ligands modulate gene expression via the translational apparatus, we take a combined structure-function approach: This entails a systematic characterization of regulatory systems using purified components in vitro to form homogenous functional complexes for structural analysis using X-ray crystallography, and also collaboratively via cryo-EM. Current work focuses on formation of ribosomal functional complexes to specifically investigate protein factors associated with stress response and ribosome biogenesis, as well as novel antimicrobial inhibitors of translation.

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