Structure and Function of the Acidic Ribosomal Stalk Proteins

Abstract

The acidic L7/L12 (prokaryotes) and P1/P2 (eukaryotes) proteins are the only ribosomal components that occur in more than one, specifically four, copies in the translational machinery. These ribosomal proteins are the only ones that do not directly interact with ribosomal RNA but bind to the particles via a protein, L10 and P0, respectively. They constitute a morphologically distinct feature on the large subunit, the stalk protuberance. Since a long time proteins L7/L12 have been implicated in translation factor binding and in the stimulation of the factor-dependent GTP-hydrolysis. Recent studies reproduced such activities with the isolated components and L7/L12 can therefore in retrospect be regarded as the first GTPase activating proteins identified. GTP-hydrolysis induces a drastic conformational change in elongation factor (EF) Tu, which enables it to dissociate from the ribosome after having successfully delivered aminoacylated tRNA into the A-site. It is also used as a driving force for translocation, mediated by EF-G. The in vitro stimulation of translation-uncoupled EF-G-dependent GTP-hydrolysis seems to be an intrinsic property of the ribosome that is dependent on L7/L12, reaches a maximum with four copies of the proteins per particle, and reflects the in vivo hydrolysis rate during translation. It is much larger than the analogous activity observed for EF-Tu, which is correlated with the in vitro polypeptide synthesis rate. Therefore, at least certain stimulatory activities of L7/L12 are controlled by the ribosomal environment, which in the case of EF-Tu senses the successful codon-anticodon pairing. Present knowledge is consistent with a picture in which proteins L7/L12 constitute a "landing platform" for the factors and after rearrangements induce GTP-hydrolysis. The molecular mechanism of the GTPase activation is unknown. While sequence comparisons show a large diversity in the stalk proteins across the kingdoms, a conserved functional domain organization and conserved designs of their genetic units are discernible. Consistently, stalk transplantation experiments suggest that coevolution took place to maintain functional L7/L12 EF-G and P-protein EF-2 couples. The acidic proteins are organized into three distinct functional parts: An N-terminal domain is responsible for oligomerization and ribosome association, a C-terminal domain is implicated in translation factor interactions, and a hinge region allows a flexible relative orientation of the latter two portions. The bacterial L7/L12 proteins have long been portrayed as highly elongated dimers displaying globular C-terminal domains, helical N-termini, and unstructured hinges. Conversely, recent crystal structures depict a compact hetero-tetrameric assembly with the hinge region adopting either an alpha-helical or an open conformation. Two different dimerization modes can be discerned in these structures. Models suggest that dimerization via one association mode can lead to elongated dimeric complexes with one helical and one unstructured hinge. The ...

The puzzling lateral flexible stalk of the ribosome

“…This is very unlikely, especially since this fold corresponds to that of the L7/L12 dimer found in the 70S ribosome (Yusupov et al, 2001). Another possibility is that the observed fold corre-sponds to a relaxed state of L7/L12, and that another conformation exists in which the hinge is extended after a helix-coil transition (hypothesis developed in (Wahl and Moller, 2002)). This would explain the different aspects of this region under the different steps of the elongation cycle Frank and Agrawal, 2000;Valle et al, 2002).…”

Archaeal ribosomal stalk protein interacts with translation factors in a nucleotide-independent manner via its conserved C terminus

“…The GTPase-associated center plays a crucial role in the recruitment of translation factors, GTP hydrolysis, and the release of inorganic phosphate, which is required for the subsequent dissociation of the factor. Multiple copies of the acidic ribosomal protein, or so-called stalk protein, are key components of this functional center (5)(6)(7)(8)(9). The stalk proteins form homo-or heterodimers, and two or three dimers bind to the ribosome through an anchor protein, L10 in bacteria and P0 in eukaryotes (7,(9)(10)(11)(12).…”

Evolution of the protein stoichiometry in the L12 stalk of bacterial and organellar ribosomes

“…The only r-protein that is present in multiple copies is L12, a protein of the large ribosomal subunit, which is part of the so-called L12 stalk of the ribosome (in bacteria and archaea, or P-stalk in eukaryotes). The L12 stalk entails ribosomal protein L10 (or its archaeal and eukaryotic orthologue P0 in the P-stalk) and multiple copies of protein L12 (P1/P2 in eukaryotes 1 ). The L12 stalk is required for translation, because it recruits to the ribosome the auxiliary translation factors, in particular translational GTPases, such as initiation factor 2, elongation factors Tu and G, and release factor 3 or their eukaryotic homologues [2][3][4][5][6][7][8] , and accelerates GTPase activity of some of them 3,[9][10][11] .…”

“…L12 entails two domains, the amino-terminal domain (NTD) and the carboxy-terminal domain, which are connected by a flexible hinge region 24 . The globular carboxy-terminal domain interacts with translation factors 1 , whereas the a-helical NTD is responsible for L12 dimerization and binding to L10. L10 contains eight a-helices and four b-sheets, and the L12 NTD dimers bind to consecutive elements in the C-terminal helix a8 of L10, which comprises a-helical segments separated by bends 9 (Fig.…”

“…From the amounts of L12 and L10 peptides, the stoichiometry L12:L10 was calculated. As a control, E. coli ribosomes were used for which the L12 copy number is known 1,9,21 .…”