A single-headed dimer of Escherichia coli ribosomal protein L7/L12 supports protein synthesis

Abstract

During protein synthesis, the two elongation factors Tu and G alternately bind to the 50S ribosomal subunit at a site of which the protein L7͞L12 is an essential component. L7͞L12 is present in each 50S subunit in four copies organized as two dimers. Each dimer consists of distinct domains: a single N-terminal (''tail'') domain that is responsible for both dimerization and binding to the ribosome via interaction with the protein L10 and two independent globular C-terminal domains (''heads'') that are required for binding of elongation factors to ribosomes. The two heads are connected by f lexible hinge sequences to the N-terminal domain. Important questions concerning the mechanism by which L7͞L12 interacts with elongation factors are posed by us in response to the presence of two dimers, two heads per dimer, and their dynamic, mobile properties. In an attempt to answer these questions, we constructed a single-headed dimer of L7͞L12 by using recombinant DNA techniques and chemical cross-linking. This chimeric molecule was added to inactive core particles lacking wild-type L7͞L12 and shown to restore activity to a level approaching that of wild-type two-headed L7͞L12.The ribosomal protein L7͞L12 is present on the ribosome in four copies as two dimers and is required for the binding of translational factors. The association between these ribosomal proteins and factors to produce GTP hydrolysis-derived energy for template-guided movement of the ribosome is of interest with respect to the mechanism of protein synthesis on the ribosome and as an example of a mechanochemical system (1), or molecular motor, and as an effector G protein system.The unique quaternary structure of L7͞L12 has been conserved in eubacteria, eukaryotes, and archea (2, 3). Protein L7͞L12 has been studied in great detail because of the ease with which it can be removed selectively from the large ribosomal subunits. The removal of L7͞L12 reduces the rate of protein synthesis by an order of magnitude and its accuracy (4, 5). Wild-type L7͞L12 can be replaced easily by variant polypeptides created by chemical or proteolytic cleavage, chemical modification, or recombinant DNA techniques as long as the ribosome binding domain is intact.Detailed structures of both organized domains [the globular C-terminal domain (residues 53-120) from x-ray crystallography (6) and the helical N-terminal dimerization domain (residues 1-37) by NMR (7)] have been determined. The two organized domains are connected by a flexible hinge sequence.

Showing your ID: intrinsic disorder as an ID for recognition, regulation and cell signaling

“…The Cterminal domain of L7/L12 has been shown by fluorescence studies to be flexible (Hamman et al, 1996a,b). This dynamic behavior may enable L7/L12 to interact with the diverse set of auxiliary translation factors that bind at the base of the stalk and guide the ribosome through the translational cycle (Oleinikov et al, 1998). In agreement with this suggestion, it has been shown recently that the Cterminal domain of L7/L12 is responsible for elongationfactor Tu (EF-Tu) function (Terasaki et al, 2004).…”

Specialized ribosomes: a new frontier in gene regulation and organismal biology

“…These findings also raise the possibility that some ribosomes may contain more than one copy of certain, highly expressed ribosomal proteins. This is analogous to the dimerization of the acidic ribosomal proteins in the ribosomes of Escherichia coli , which contain two protein copies of RpL7 and RpL12 per ribosome 43-45 . Importantly, as ribosomal proteins may have extra-ribosomal functions, can be degraded at the protein level 46 or are translationally controlled 146 , further proteomics studies will be key in confirming the contribution of heterogenous ribosomal protein expression in specialized ribosomal activity.…”

Heteronuclear NMR investigations of dynamic regions of intact Escherichia coli ribosomes

“…The L7͞L12 proteins are crucial for accurate translation of mRNA (32), and many biophysical studies of L7͞L12 have been carried out to probe the mode of action of these molecules (33). The CTD of L7͞L12 has been shown by fluorescence studies (34,35) to be flexible; such motion is thought to enable L7͞L12 to interact with the diverse set of auxiliary translation factors that bind at the base of the stalk and guide the ribosome through the translational cycle (36). Indeed, very recent evidence strongly suggests that the CTD of L7͞L12 is responsible for elongation-factor Tu (EF-Tu) function (37).…”