The pathogen protein EspF(U) hijacks actin polymerization using mimicry and multivalency

Enterohaemorrhagic Escherichia coli attaches to the intestine through actin pedestals that are formed when the bacterium injects its protein EspF(U) (also known as TccP) into host cells. EspF(U) potently activates the host WASP (Wiskott-Aldrich syndrome protein) family of actin-nucleating factors, which are normally activated by the GTPase CDC42, among other signalling molecules. Apart from its amino-terminal type III secretion signal, EspF(U) consists of five-and-a-half 47-amino-acid repeats. Here we show that a 17-residue motif within this EspF(U) repeat is sufficient for interaction with N-WASP (also known as WASL). Unlike most pathogen proteins that interface with the cytoskeletal machinery, this motif does not mimic natural upstream activators: instead of mimicking an activated state of CDC42, EspF(U) mimics an autoinhibitory element found within N-WASP. Thus, EspF(U) activates N-WASP by competitively disrupting the autoinhibited state. By mimicking an internal regulatory element and not the natural activator, EspF(U) selectively activates only a precise subset of CDC42-activated processes. Although one repeat is able to stimulate actin polymerization, we show that multiple-repeat fragments have notably increased potency. The activities of these EspF(U) fragments correlate with their ability to coordinate activation of at least two N-WASP proteins. Thus, this pathogen has used a simple autoinhibitory fragment as a component to build a highly effective actin polymerization machine.     

Multi-domain conformational selection underlies pre-mRNA splicing regulation by U2AF

Many cellular functions involve multi-domain proteins, which are composed of structurally independent modules connected by flexible linkers. Although it is often well understood how a given domain recognizes a cognate oligonucleotide or peptide motif, the dynamic interaction of multiple domains in the recognition of these ligands remains to be characterized. Here we have studied the molecular mechanisms of the recognition of the 3'-splice-site-associated polypyrimidine tract RNA by the large subunit of the human U2 snRNP auxiliary factor (U2AF65) as a key early step in pre-mRNA splicing. We show that the tandem RNA recognition motif domains of U2AF65 adopt two remarkably distinct domain arrangements in the absence or presence of a strong (that is, high affinity) polypyrimidine tract. Recognition of sequence variations in the polypyrimidine tract RNA involves a population shift between these closed and open conformations. The equilibrium between the two conformations functions as a molecular rheostat that quantitatively correlates the natural variations in polypyrimidine tract nucleotide composition, length and functional strength to the efficiency to recruit U2 snRNP to the intron during spliceosome assembly. Mutations that shift the conformational equilibrium without directly affecting RNA binding modulate splicing activity accordingly. Similar mechanisms of cooperative multi-domain conformational selection may operate more generally in the recognition of degenerate nucleotide or amino acid motifs by multi-domain proteins.     

酵母snoRNA基因簇启动子的比较分析

报道对酿酒酵母ｓｎｏＲＮＡ基因簇启动子序列的研究结果．通过计算机分析,发现酿酒酵母中Ｚ２Ｚ８和ＳｎＲ１９０Ｕ１４ｓｎｏＲＮＡ基因簇有相似的启动子结构．这两个ｓｎｏＲＮＡ基因簇都是由一个上游的启动子负责整个基因簇的转录,产生多个顺反子ｓｎｏＲＮＡ的前体,然后再加工成熟为各个独立的ｓｎｏＲＮＡ．在启动子保守序列ＴＡＴＡｂｏｘ的上游还发现２个ＲＡＰ１序列,表明ｓｎｏＲＮＡ基因簇的表达可以通过ＲＡＰ１元素与核糖体蛋白基因的表达协同调控．这是在ｓｎｏＲＮＡ基因的启动子中首次发现ＲＡＰ１调控元素．对ｓｎｏＲＮＡ基因簇的进化特点也进行了讨论．     

Internal sequences that distinguish yeast from metazoan U2 snRNA are unnecessary for pre-mRNA splicing

U2 small nuclear RNA is a highly conserved component of the eukaryotic cell nucleus involved in splicing messenger RNA precursors. In the yeast Saccharomyces cerevisiae, U2 RNA interacts with the intron by RNA-RNA pairing between the conserved branchpoint sequence UACUAAC and conserved nucleotides near the 5' end of U2 (ref. 4). Metazoan U2 RNA is less than 200 nucleotides in length, but yeast U2 RNA is 1,175 nucleotides long. The 5' 110 nucleotides of yeast U2 are homologous to the 5' 100 nucleotides of metazoan U2 (ref. 6), and the very 3' end of yeast U2 bears a weak structural resemblance to features near the 3' end of metazoan U2. Internal sequences of yeast U2 share primary sequence homology with metazoan U4, U5 and U6 small nuclear RNA (ref. 6), and have regions of complementarity with yeast U1 (ref. 7). We have investigated the importance of the internal U2 sequences by their deletion. Yeast cells carrying a U2 allele lacking 958 nucleotides of internal U2 sequence produce a U2 small nuclear RNA similar in size to that found in other organisms. Cells carrying only the U2 deletion grow normally, have normal levels of spliced mRNA and do not accumulate unspliced precursor mRNA. We conclude that the internal sequences of yeast U2 carry no essential function. The extra RNA may have a non-essential function in efficient ribonucleoprotein assembly or RNA stability. Variation in amount of RNA in homologous structural RNAs has precedence in ribosomal RNA and RNaseP.     

RNA polymerase II C-terminal repeat influences response to transcriptional enhancer signals

The large subunit of RNA polymerase II contains a highly conserved and essential heptapeptide repeat (Pro-Thr-Ser-Pro-Ser-Tyr-Ser) at its carboxy terminus. Saccharomyces cerevisiae cells are inviable if their RNA polymerase II large subunit genes encode fewer than 10 complete heptapeptide repeats; if they encode 10 to 12 complete repeats cells are temperature-sensitive and cold-sensitive, but 13 or more complete repeats will allow wild-type growth at all temperatures. Cells containing C-terminal domains (CTDs) of 10 to 12 complete repeats are also inositol auxotrophs. The phenotypes associated with these CTD mutations are not a consequence of an instability of the large subunit; rather, they seem to reflect a functional deficiency of the mutant enzyme. We show here that partial deletion mutations in RNA polymerase II CTD affect the ability of the enzyme to respond to signals from upstream activating sequences in a subset of promoters in yeast. The number of heptapeptide repeats required for maximal response to signals from these sequences differs from one upstream activating sequence to another. One of the upstream elements that is sensitive to truncations of the CTD is the 17-base-pair site bound by the GAL4 transactivating factor.     

Rotavirus protein involved in genome replication and packaging exhibits a HIT-like fold

Rotavirus, the major cause of life-threatening infantile gastroenteritis, is a member of the Reoviridae. Although the structures of rotavirus and other members of the Reoviridae have been extensively studied, little is known about the structures of virus-encoded non-structural proteins that are essential for genome replication and packaging. The non-structural protein NSP2 of rotavirus, which exhibits nucleoside triphosphatase, single-stranded RNA binding, and nucleic-acid helix-destabilizing activities, is a major component of viral replicase complexes. We present here the X-ray structure of the functional octamer of NSP2 determined to a resolution of 2.6 A. The NSP2 monomer has two distinct domains. The amino-terminal domain has a new fold. The carboxy-terminal domain resembles the ubiquitous cellular histidine triad (HIT) group of nucleotidyl hydrolases. This structural similarity suggests that the nucleotide-binding site is located inside the cleft between the two domains. Prominent grooves that run diagonally across the doughnut-shaped octamer are probable locations for RNA binding. Several RNA binding sites, resulting from the quaternary organization of NSP2 monomers, may be required for the helix destabilizing activity of NSP2 and its function during genome replication and packaging.