Ribosomal Stalk Protein Research Articles

Conformation and dynamics of ribosomal stalk protein L12 in solution and on the ribosome.

During translation, the ribosome and several of its constituent proteins undergo structural transitions between different functional states. Protein L12, present in four copies in prokaryotic ribosomes, forms a flexible "stalk" with key functions in factor-dependent GTP hydrolysis during translocation. Here we have used heteronuclear NMR spectroscopy to characterize L12 conformation and dynamics in solution and on the ribosome. Isolated L12 forms a symmetric dimer mediated by the N-terminal domains (NTDs), to which each C-terminal domain (CTD) is connected via an unstructured hinge segment. The overall structure can be described as three ellipsoids joined by flexible linkers. No persistent contacts are seen between the two CTDs, or between the NTD and CTD in the L12 dimer in solution. In the (1)H-(15)N HSQC spectrum of the Escherichia coli 70S ribosome, a single set of cross-peaks are observed for residues 40-120 of L12, the intensities of which correspond to only two of four protein copies. The structure of the CTDs observed on the ribosome is indistinguishable from that of isolated L12. These results indicate that two CTDs with identical average structures are mobile and extend away from the ribosome, while the other two copies most likely interact tightly with the ribosome even in the absence of translational factors.

Protein BmP0 from the silkworm Bombyx mori can be assembled and is functional in the Saccharomyces cerevisiae ribosomal stalk in the absence of the acidic P1 and P2 proteins

The DNA complementary to RNA (cDNA) of the ribosomal stalk protein BmP0 of the silkworm Bombyx mori was isolated from a cDNA library and was subsequently expressed in the conditional P0-null mutant Saccharomyces cerevisiae D67dGP0, whose ribosomes also lack the other stalk components, proteins P1/P2. The transformed strain was able to grow under restrictive conditions, indicating that in the absence of the P1/P2 proteins BmP0 can bind to the yeast ribosomes and complement the lack of the endogenous YP0 protein . In addition, the binding capacity of the B. mori ribosomal stalk components to the ribosomal particle was studied by means of high salt treatment of purified ribosomes. The BmP0 protein retained its binding to the ribosome, suggesting a stable association with the rRNA, in contrast to the acidic proteins BmP1 and BmP2, which were easily released. The results clearly indicate that, as opposed to recent in vitro results, BmP0 does not require the presence of P1/P2 proteins in order to bind to the ribosome.

Structure and function of the acidic ribosomal stalk proteins.

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 physiological role of the other dimerization mode is unclear and is in apparent contradiction to distances measured by fluorescence resonance energy transfer. The discrepancies between the crystal structures and results from other physico-chemical methods may partly be a consequence of the dynamic functions of the proteins, necessitating a high flexibility.

Species-specific inhibition of fungal protein synthesis by sordarin: identification of a sordarin-specificity region in eukaryotic elongation factor 2.

The sordarin class of natural products selectively inhibits fungal protein synthesis by impairing the function of eukaryotic elongation factor 2 (eEF2). Mutations in Saccharomyces cerevisiae eEF2 or the ribosomal stalk protein rpP0 can confer resistance to sordarin, although eEF2 is the major determinant of sordarin specificity. It has been shown previously that sordarin specifically binds S. cerevisiae eEF2 while there is no detectable binding to eEF2 from plants or mammals, despite the high level of amino acid sequence conservation among these proteins. In both whole-cell assays and in vitro translation assays, the efficacy of sordarin varies among different species of pathogenic fungi. To investigate the basis of sordarin's fungal selectivity, eEF2 has been cloned and characterized from several sordarin-sensitive and -insensitive fungal species. Results from in vivo expression of Candida species eEF2s in S. cerevisiae and in vitro translation and growth inhibition assays using hybrid S. cerevisiae eEF2 proteins demonstrate that three amino acid residues within eEF2 account for the selectivity of this class of compounds. It is also shown that the corresponding residues at these positions in human eEF2 are sufficient to confer sordarin insensitivity to S. cerevisiae identical to that observed with mammalian eEF2.

Structure function relationships in the ribosomal stalk proteins of archaebacteria.

The ribosomal L12 protein gene of Sulfolobus solfataricus (SsoL12) has been subcloned and overexpressed in Escherichia coli. Five protein L12 mutants were designed: two NH2-terminal and two COOH-terminal truncated mutants and one mutant lacking the highly charged part of the COOH-terminal region. The mutant protein genes were overexpressed in E. coli and the products purified. The amino acid composition was verified and the NH2 terminally truncated mutants were subjected to Edman degradation. The SsoL12 protein was selectively removed from entire S. solfataricus ribosomes by an ethanol wash. The remaining ribosomal core particles showed a substantial decrease in the in vitro translational activity. S. solfataricus L12 protein overexpressed in E. coli (SsoL12e) was incorporated into these ribosomal cores and restored their translational activity. Mutants lacking any part of the COOH-terminal region could be incorporated into these cores, as proven by two-dimensional polyacrylamide gels of the reconstituted particles. Mutant SsoL12 MC2 (residue 1-70) was sufficient for dimerization and incorporation into ribosomes. In contrast to the COOH terminally truncated mutants, L12 proteins lacking the 12 highly conserved NH2-terminal residues or the entire NH2-terminal region (44 amino acids) are unable to bind to ribosomes, suggesting that the SsoL12 protein binds with its NH2-terminal portion to the ribosome. None of the mutants could significantly increase the translational activity of the core particles suggesting that every deleted part of the protein was needed directly or indirectly for translational activity. Our results suggest that the COOH terminally truncated mutants were bound to ribosomes but not functional for translation. Cores preincubated with these COOH terminally truncated mutants regained activity when a second incubation with the entire overexpressed SsoL12e protein followed. This finding suggests that archaebacterial L12 proteins are freely exchanged on the ribosome.