Structural visualization of key steps in human transcription initiation.

Eukaryotic transcription initiation requires the assembly of general transcription factors into a pre-initiation complex that ensures the accurate loading of RNA polymerase II (Pol II) at the transcription start site. The molecular mechanism and function of this assembly have remained elusive due to lack of structural information. Here we have used an in vitro reconstituted system to study the stepwise assembly of human TBP, TFIIA, TFIIB, Pol II, TFIIF, TFIIE and TFIIH onto promoter DNA using cryo-electron microscopy. Our structural analyses provide pseudo-atomic models at various stages of transcription initiation that illuminate critical molecular interactions, including how TFIIF engages Pol II and promoter DNA to stabilize both the closed pre-initiation complex and the open-promoter complex, and to regulate start--initiation complexes, combined with the localization of the TFIIH helicases XPD and XPB, support a DNA translocation model of XPB and explain its essential role in promoter opening

Regulation of Murine Class I Genes by Interferons is Controlled by Regions Located Both 5' and 3' to the Transcription Initiation Site

Interferons regulate the expression of a large number of mammalian genes, including the major histocompatibility antigen genes. To investigate the mechanisms involved in interferon action, we have analyzed the ability of murine H-2Ld and H-2Dd DNA sequences to control the responses to interferon. The results indicate that interferon regulation of class I gene expression is complex and involves at least two mechanisms that are dependent on class I sequences located upstream and downstream to the transcription initiation site. In transfected mouse L cells, both of these regions are required for full enhancement of class I gene expression, with the major portion of the response controlled by the sequences located 3' to the transcription initiation site. The fine-mapping analysis of the 5' region-encoded response also suggests that recombinant alpha and gamma interferons may exert their effects on class I gene expression by using different cis-acting regulatory sequences

Ribosome signatures aid bacterial translation initiation site identification

Background: While methods for annotation of genes are increasingly reliable, the exact identification of translation initiation sites remains a challenging problem. Since the N-termini of proteins often contain regulatory and targeting information, developing a robust method for start site identification is crucial. Ribosome profiling reads show distinct patterns of read length distributions around translation initiation sites. These patterns are typically lost in standard ribosome profiling analysis pipelines, when reads from footprints are adjusted to determine the specific codon being translated. Results: Utilising these signatures in combination with nucleotide sequence information, we build a model capable of predicting translation initiation sites and demonstrate its high accuracy using N-terminal proteomics. Applying this to prokaryotic translatomes, we re-annotate translation initiation sites and provide evidence of N-terminal truncations and extensions of previously annotated coding sequences. These re-annotations are supported by the presence of structural and sequence-based features next to N-terminal peptide evidence. Finally, our model identifies 61 novel genes previously undiscovered in the Salmonella enterica genome. Conclusions: Signatures within ribosome profiling read length distributions can be used in combination with nucleotide sequence information to provide accurate genome-wide identification of translation initiation sites

Translation initiation from the ribosomal A site or the P site, dependent on the conformation of RNA pseudoknot I in dicistrovirus RNAs

available in PMC 2010 July 31.Translation initiation of the second ORF of insect dicistrovirus RNA depends on an internal ribosomal entry site (IRES) in its intergenic region (IGR) and is exceptional in using a codon other than AUG and in not using the canonical initiator methionine tRNA. Studies in vitro suggest that pseudoknot I (PKI) immediately preceding the initiation codon occupies the ribosomal P site and that an elongator tRNA initiates translation from the ribosomal A site. Using dicistronic reporters carrying mutations in the initiation codon of the second ORF and mutant elongator or initiator tRNAs capable of reading these codons, we provide direct evidence for initiation from the A site in mammalian cells and, under certain conditions, also from the P site. Initiation from the A but not the P site requires PKI. Thus, PKI structure may be dynamic, and optimal IGR IRES-mediated translation of dicistroviral RNAs may require trans-acting factors to stabilize PKI.National Institutes of Health (U.S.) (grant GM17151)Japan. Ministry of Education, Culture, Sports, Science and Technology (Grants-In-Aid for Scientific Research on Priority Areas

A nucleosome-free dG-dC-rich sequence element promotes constitutive transcription of the essential yeast RIO1 gene

RIO1 is an essential gene that encodes a protein serine kinase and is transcribed constitutively at a very low level. Transcriptional activation of RIO1 dispenses with a canonical TATA box as well as with classical transactivators or specific DNAbinding factors. Instead, a dGdCrich sequence element, that is located 40 to 48 bp upstream the single site of mRNA initiation, is essential and presumably constitutes the basal promoter. In addition, we demonstrate here that this promoter element comprises a nucleosomefree gap which is centered at the dGdC tract and flanked by two positioned nucleosomes. This element is both, necessary and sufficient, for basal transcription initiation at the RIO1 promoter and, thus, constitutes a novel type of core promoter element

A Pan1/End3/Sla1 complex links Arp2/3-mediated actin assembly to sites of clathrin-mediated endocytosis.

More than 60 highly conserved proteins appear sequentially at sites of clathrin-mediated endocytosis in yeast and mammals. The yeast Eps15-related proteins Pan1 and End3 and the CIN85-related protein Sla1 are known to interact with each other in vitro, and they all appear after endocytic-site initiation but before endocytic actin assembly, which facilitates membrane invagination/scission. Here we used live-cell imaging in parallel with genetics and biochemistry to explore comprehensively the dynamic interactions and functions of Pan1, End3, and Sla1. Our results indicate that Pan1 and End3 associate in a stable manner and appear at endocytic sites before Sla1. The End3 C-terminus is necessary and sufficient for its cortical localization via interaction with Pan1, whereas the End3 N-terminus plays a crucial role in Sla1 recruitment. We systematically examined the dynamic behaviors of endocytic proteins in cells in which Pan1 and End3 were simultaneously eliminated, using the auxin-inducible degron system. The results lead us to propose that endocytic-site initiation and actin assembly are separable processes linked by a Pan1/End3/Sla1 complex. Finally, our study provides mechanistic insights into how Pan1 and End3 function with Sla1 to coordinate cargo capture with actin assembly

Two RNA-binding motifs in eIF3 direct HCV IRES-dependent translation.

The initiation of protein synthesis plays an essential regulatory role in human biology. At the center of the initiation pathway, the 13-subunit eukaryotic translation initiation factor 3 (eIF3) controls access of other initiation factors and mRNA to the ribosome by unknown mechanisms. Using electron microscopy (EM), bioinformatics and biochemical experiments, we identify two highly conserved RNA-binding motifs in eIF3 that direct translation initiation from the hepatitis C virus internal ribosome entry site (HCV IRES) RNA. Mutations in the RNA-binding motif of subunit eIF3a weaken eIF3 binding to the HCV IRES and the 40S ribosomal subunit, thereby suppressing eIF2-dependent recognition of the start codon. Mutations in the eIF3c RNA-binding motif also reduce 40S ribosomal subunit binding to eIF3, and inhibit eIF5B-dependent steps downstream of start codon recognition. These results provide the first connection between the structure of the central translation initiation factor eIF3 and recognition of the HCV genomic RNA start codon, molecular interactions that likely extend to the human transcriptome

Hepatitis C virus 3'UTR regulates viral translation through direct interactions with the host translation machinery.

The 3' untranslated region (3'UTR) of hepatitis C virus (HCV) messenger RNA stimulates viral translation by an undetermined mechanism. We identified a high affinity interaction, conserved among different HCV genotypes, between the HCV 3'UTR and the host ribosome. The 3'UTR interacts with 40S ribosomal subunit proteins residing primarily in a localized region on the 40S solvent-accessible surface near the messenger RNA entry and exit sites. This region partially overlaps with the site where the HCV internal ribosome entry site was found to bind, with the internal ribosome entry site-40S subunit interaction being dominant. Despite its ability to bind to 40S subunits independently, the HCV 3'UTR only stimulates translation in cis, without affecting the first round translation rate. These observations support a model in which the HCV 3'UTR retains ribosome complexes during translation termination to facilitate efficient initiation of subsequent rounds of translation

Mapping replication dynamics in Trypanosoma brucei reveals a link with telomere transcription and antigenic variation

Survival of Trypanosoma brucei depends upon switches in its protective Variant Surface Glycoprotein (VSG) coat by antigenic variation. VSG switching occurs by frequent homologous recombination, which is thought to require locus-specific initiation. Here, we show that a RecQ helicase, RECQ2, acts to repair DNA breaks, including in the telomeric site of VSG expression. Despite this, RECQ2 loss does not impair antigenic variation, but causes increased VSG switching by recombination, arguing against models for VSG switch initiation through direct generation of a DNA double strand break (DSB). Indeed, we show DSBs inefficiently direct recombination in the VSG expression site. By mapping genome replication dynamics, we reveal that the transcribed VSG expression site is the only telomeric site that is early replicating – a differential timing only seen in mammal-infective parasites. Specific association between VSG transcription and replication timing reveals a model for antigenic variation based on replication-derived DNA fragility

Surface characterization and influence of anodizing process on fatigue life of Al 7050 alloy

The present study investigates the influence of anodizing process on fatigue life of aluminium alloy 7050-T7451 by performing axial fatigue tests at stress ratio ‘R’ of 0.1. Effects of pre-treatments like degreasing and pickling employed prior to anodizing on fatigue life were studied. The post-exposure surface observations were made by scanning electron microscope (SEM) to characterize the effect of each treatment before fatigue testing. The surface observations have revealed that degreasing did not change the surface topography while pickling solution resulted in the formation of pits at the surface. Energy dispersive spectroscopy (EDS) was used to identify those constituent particles which were responsible for the pits formation. These pits are of primary concern with respect to accelerated fatigue crack initiation and subsequent anodic coating formation. The fatigue test results have shown that pickling process was detrimental in reducing the fatigue life significantly while less decrease has been observed for anodized specimens. Analyses of fracture surfaces of pickled specimens have revealed that the process completely changed the crack initiation mechanisms as compared to non-treated specimens and the crack initiation started at the pits. For most of the anodized specimens, fatigue cracks still initiated at the pits with very few cracks initiated from anodic coating. The decrease in fatigue life for pickled and anodized specimens as compared to bare condition has been attributed to decrease in initiation period and multi-site crack initiations. Multi-site crack initiation has resulted in rougher fractured surfaces for the pickled and anodized specimens as compare to bare specimens tested at same stress levels