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The yeast Vts1 SAM (sterile alpha motif) domain is a member of a new class of SAM domains that specifically bind RNA. To elucidate the structural basis for RNA binding, the solution structure of the Vts1 SAM domain, in the presence of a specific target RNA, has been solved by multidimensional heteronuclear NMR spectroscopy. The Vts1 SAM domain retains the "core" five-helix-bundle architecture of traditional SAM domains, but has additional short helices at N and C termini, comprising a small substructure that caps the core helices. The RNA-binding surface of Vts1, determined by chemical shift perturbation, maps near the ends of three of the core helices, in agreement with mutational data and the electrostatic properties of the molecule. These results provide a structural basis for the versatility of the SAM domain in protein and RNA-recognition.  相似文献   

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In Saccharomyces cerevisiae, the MAPKKK Ste11p is involved in three mitogen-activated protein kinase (MAPK) pathways required for mating, filamentous growth and the SHO1-dependent response to hyperosmolarity. All three pathways are also dependent on Ste50p. Ste50p and Ste11p interact constitutively via their N-terminal regions, which include putative SAM domains. Here we show that the interaction of Ste50p and Ste11p is differentially required for modulation of Ste11p function during mating, filamentous growth and the SHO1-dependent response to hyperosmolarity. Two derivatives of Ste50p with mutations in the SAM domain were isolated and characterised. The mutant Ste50 proteins showed reduced binding to Ste11p and a tendency to form homodimers in two-hybrid and in vitro binding assays. Interestingly, these two Ste50p-SAM mutants were associated with increased activation of the mating and filamentous-growth pathways, but a reduction in the SHO1-dependent growth response to hyperosmolarity, relative to the wild-type Ste50p. Moreover, when exposed to hyperosmolarity, these Ste50p-SAM mutants activate genes in the mating (FUS1) and filamentous-growth (FLO11) pathways to higher levels than does the wild type. Thus the Ste50p-Ste11p interaction may differentially modulate the flow of information through the various MAPK-mediated pathways.  相似文献   

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p63 is a member of the p53 tumour suppressor family that includes p73. The p63 gene encodes a protein comprising an N-terminal transactivation domain, a DNA binding domain and an oligomerization domain, but varies in the organization of the C-terminus as a result of complex alternative splicing. p63α contains a C-terminal sterile α motif (SAM) domain that is thought to function as a protein-protein interaction domain. Several missense and heterozygous frame shift mutations, encoded within exon 13 and 14 of the p63 gene, have been identified in the p63α SAM domain in patients suffering from ankyloblepharon-ectodermal dysplasia-clefting syndrome. Here we report the solution and high resolution crystal structures of the p63α SAM domain and investigate the effect of several mutations (L553F/V, C562G/W, G569V, Q575L and I576T) on the stability of the domain. The possible effects of other mutations are also discussed.  相似文献   

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The essential Saccharomyces cerevisiae PRP22 gene encodes a 1145-amino acid DEXH box RNA helicase. Prp22p plays two roles during pre-mRNA splicing as follows: it is required for the second transesterification step and for the release of mature mRNA from the spliceosome. Whereas the step 2 function of Prp22p does not require ATP hydrolysis, spliceosome disassembly is dependent on the ATPase and helicase activities. Here we delineate a minimal functional domain, Prp22(262-1145), that suffices for the activity of Prp22p in vivo when expressed under the natural PRP22 promoter and for pre-mRNA splicing activity in vitro. The biologically active domain lacks an S1 motif (residues 177-256) that had been proposed to play a role in RNA binding by Prp22p. The deletion mutant Prp22(351-1145) can function in vivo when provided at a high gene dosage. We suggest that the segment from residues 262 to 350 enhances Prp22p function in vivo, presumably by targeting Prp22p to the spliceosome. We characterize an even smaller catalytic domain, Prp22(466-1145) that suffices for ATP hydrolysis, RNA binding, and RNA unwinding in vitro and for nuclear localization in vivo but cannot by itself support cell growth. However, the ATPase/helicase domain can function in vivo if the N-terminal region Prp22(1-480) is co-expressed in trans.  相似文献   

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Structural evolution of C-terminal domains in the p53 family   总被引:1,自引:0,他引:1  
Ou HD  Löhr F  Vogel V  Mäntele W  Dötsch V 《The EMBO journal》2007,26(14):3463-3473
The tetrameric state of p53, p63, and p73 has been considered one of the hallmarks of this protein family. While the DNA binding domain (DBD) is highly conserved among vertebrates and invertebrates, sequences C-terminal to the DBD are highly divergent. In particular, the oligomerization domain (OD) of the p53 forms of the model organisms Caenorhabditis elegans and Drosophila cannot be identified by sequence analysis. Here, we present the solution structures of their ODs and show that they both differ significantly from each other as well as from human p53. CEP-1 contains a composite domain of an OD and a sterile alpha motif (SAM) domain, and forms dimers instead of tetramers. The Dmp53 structure is characterized by an additional N-terminal beta-strand and a C-terminal helix. Truncation analysis in both domains reveals that the additional structural elements are necessary to stabilize the structure of the OD, suggesting a new function for the SAM domain. Furthermore, these structures show a potential path of evolution from an ancestral dimeric form over a tetrameric form, with additional stabilization elements, to the tetramerization domain of mammalian p53.  相似文献   

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The complexity of eukaryotic mRNA processing suggests a need for certain factors, called RNA chaperones, that can modulate RNA secondary structure as well as the interactions between pre-mRNA and trans-acting components. To identify factors that may fulfill this role in the yeast Saccharomyces cerevisiae, we fractionated whole-cell extracts and assayed for activity that could facilitate a specific RNA-RNA annealing reaction. We detected one strong RNA annealing activity and purified it to homogeneity. This previously undescribed factor, Yra1p, is localized to the nucleus; its sequence contains one RNP-motif RNA-binding domain. The YRA1 gene contains a 766-nt intron, the second-largest identified in this organism, and Yra1p serves an essential, nonredundant function. Taken together, our findings indicate that Yra1p is likely to have an important role in S. cerevisiae nuclear pre-mRNA metabolism.  相似文献   

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In Saccharomyces cerevisiae the SAM1 and SAM2 genes encode two distinct forms of S-adenosylmethionine (AdoMet) synthetase. In a previous study we cloned and sequenced the SAM1 gene (D. Thomas and Y. Surdin-Kerjan, J. Biol. Chem. 262:16704-16709, 1987). In this work, the SAM2 gene was isolated by functional complementation of a yeast double-mutant strain, and its identity was ascertained by gene disruption. It has been sequenced and compared with the SAM1 gene. The degree of homology found between the two genes shows that SAM1 and SAM2 are duplicated genes. Using strains disrupted in one or the other SAM gene, we have studied the regulation of their expression by measuring the steady-state level of mRNA after growth under different conditions. The results show that the expression of the two SAM genes is regulated differently, SAM2 being induced by the presence of excess methionine in the growth medium and SAM1 being repressed under the same conditions. The level of mRNA in the parental strain shows that it is not the sum of the levels found in the two disrupted strains. This raises the question of how the two AdoMet synthetases in S. cerevisiae interact to control AdoMet synthesis.  相似文献   

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