Increased accommodation of nascent RNA in a product site on RNA polymerase II during arrest.

RNA polymerases encounter specific DNA sites at which RNA chain elongation takes place in the absence of enzyme translocation in a process called discontinuous elongation. For RNA polymerase II, at least some of these sequences also provoke transcriptional arrest where renewed RNA polymerization requires elongation factor SII. Recent elongation models suggest the occupancy of a site within RNA polymerase that accommodates nascent RNA during discontinuous elongation. Here we have probed the extent of nascent RNA extruded from RNA polymerase II as it approaches, encounters, and departs an arrest site. Just upstream of an arrest site, 17-19 nucleotides of the RNA 3'-end are protected from exhaustive digestion by exogenous ribonuclease probes. As RNA is elongated to the arrest site, the enzyme does not translocate and the protected RNA becomes correspondingly larger, up to 27 nucleotides in length. After the enzyme passes the arrest site, the protected RNA is again the 18-nucleotide species typical of an elongation-competent complex. These findings identify an extended RNA product groove in arrested RNA polymerase II that is probably identical to that emptied during SII-activated RNA cleavage, a process required for the resumption of elongation. Unlike Escherichia coli RNA polymerase at a terminator, arrested RNA polymerase II does not release its RNA but can reestablish the normal elongation mode downstream of an arrest site. Discontinuous elongation probably represents a structural change that precedes, but may not be sufficient for, arrest by RNA polymerase II.

Escherichia coli trigger factor is a prolyl isomerase that associates with nascent polypeptide chains.

Correct folding of newly synthesized proteins is proposed to be assisted by molecular chaperones and folding catalysts. To identify cellular factors involved in the initial stages of this process we searched for proteins associated with nascent polypeptide chains. In an Escherichia coli transcription/translation system synthesizing beta-galactosidase we identified a 58-kDa protein which associated with translating ribosomes but dissociated from these ribosomes upon release of nascent beta-galactosidase. N-terminal sequencing identified it as trigger factor, previously implicated in protein secretion. Direct evidence for association of trigger factor with nascent polypeptide chains was obtained by crosslinking. In a wheat germ translation system complemented with E. coli lysates, epsilon-4-(3-trifluoromethyldiazirino)benzoic acid-lysine residues were incorporated into nascent secretory preprolactin and a nonsecretory preprolactin mutant. Trigger factor crosslinked to both types of nascent chains, provided they were ribosome bound. Trigger factor contains key residues of the substrate-binding pocket of FK506-binding protein-type peptidyl-prolyl-cis/trans-isomerases and has prolyl isomerase activity in vitro. We propose that trigger factor is a folding catalyst acting cotranslationally.

The intrinsic ability of ribosomes to bind to endoplasmic reticulum membranes is regulated by signal recognition particle and nascent-polypeptide-associated complex.

Signal peptides direct the cotranslational targeting of nascent polypeptides to the endoplasmic reticulum (ER). It is currently believed that the signal recognition particle (SRP) mediates this targeting by first binding to signal peptides and then by directing the ribosome/nascent chain/SRP complex to the SRP receptor at the ER. We show that ribosomes can mediate targeting by directly binding to translocation sites. When purified away from cytosolic factors, including SRP and nascent-polypeptide-associated complex (NAC), in vitro assembled translation intermediates representing ribosome/nascent-chain complexes efficiently bound to microsomal membranes, and their nascent polypeptides could subsequently be efficiently translocated. Because removal of cytosolic factors from the ribosome/nascent-chain complexes also resulted in mistargeting of signalless nascent polypeptides, we previously investigated whether readdition of cytosolic factors, such as NAC and SRP, could restore fidelity to targeting. Without SRP, NAC prevented all nascent-chain-containing ribosomes from binding to the ER membrane. Furthermore, SRP prevented NAC from blocking ribosome-membrane association only when the nascent polypeptide contained a signal. Thus, NAC is a global ribosome-binding prevention factor regulated in activity by signal-peptide-directed SRP binding. A model presents ribosomes as the targeting vectors for delivering nascent polypeptides to translocation sites. In conjunction with signal peptides, SRP and NAC contribute to this specificity of ribosomal function by regulating exposure of a ribosomal membrane attachment site that binds to receptors in the ER membrane.

Peptide inhibitors of peptidyltransferase alter the conformation of domains IV and V of large subunit rRNA: a model for nascent peptide control of translation.

Peptides of 5 and 8 residues encoded by the leaders of attenuation regulated chloramphenicol-resistance genes inhibit the peptidyltransferase of microorganisms from the three kingdoms. Therefore, the ribosomal target for the peptides is likely to be a conserved structure and/or sequence. The inhibitor peptides "footprint" to nucleotides of domain V in large subunit rRNA when peptide-ribosome complexes are probed with dimethyl sulfate. Accordingly, rRNA was examined as a candidate for the site of peptide binding. Inhibitor peptides MVKTD and MSTSKNAD were mixed with rRNA phenol-extracted from Escherichia coli ribosomes. The conformation of the RNA was then probed by limited digestion with nucleases that cleave at single-stranded (T1 endonuclease) and double-stranded (V1 endonuclease) sites. Both peptides selectively altered the susceptibility of domains IV and V of 23S rRNA to digestion by T1 endonuclease. Peptide effects on cleavage by V1 nuclease were observed only in domain V. The T1 nuclease susceptibility of domain V of in vitro-transcribed 23S rRNA was also altered by the peptides, demonstrating that peptide binding to the rRNA is independent of ribosomal protein. We propose the peptides MVKTD and MSTSKNAD perturb peptidyltransferase center catalytic activities by altering the conformation of domains IV and V of 23S rRNA. These findings provide a general mechanism through which nascent peptides may cis-regulate the catalytic activities of translating ribosomes.

Nascent polypeptide-associated complex protein prevents mistargeting of nascent chains to the endoplasmic reticulum.

We show that, after removal of the nascent polypeptide-associated complex (NAC) from ribosome-associated nascent chains, ribosomes synthesizing proteins lacking signal peptides are efficiently targeted to the endoplasmic reticulum (ER) membrane. After this mistargeting, translocation across the ER membrane occurs, albeit less efficiently than for a nascent secretory polypeptide, perhaps because the signal peptide is needed to catalyze the opening of the translocation pore. The mistargeting was prevented by the addition of purified NAC and was shown not to be mediated by the signal recognition particle and its receptor. Instead, it appears to be a consequence of the intrinsic affinity of ribosomes for membrane binding sites, since it can be blocked by competing ribosomes that lack associated nascent polypeptides. We propose that, when bound to a signalless ribosome-associated nascent polypeptide, NAC sterically blocks the site in the ribosome for membrane binding.

NusA interferes with interactions between the nascent RNA and the C-terminal domain of the alpha subunit of RNA polymerase in Escherichia coli transcription complexes.

The effects of NusA on the RNA polymerase contacts made by nucleotides at internal positions in the nascent RNA in Escherichia coli transcription complexes were analyzed by using the photocrosslinking nucleotide analog 5-[(4-azidophenacyl) thio]-UMP. It was placed at nucleotides between +6 and +15 in RNA transcribed from the phage lambda PR' promoter. Crosslinks of analog in these positions in RNAs which contained either 15, 28, 29, or 49 nt were examined. Contacts between the nascent RNA and proteins in the transcription complex were analyzed as the RNA was elongated, by placing the crosslinker nearest the 5' end of the RNA 10, 23, 24, or 44 nt away from the 3' end. The beta or beta' subunit of polymerase, and NusA when added, were contacted by RNA from 15 to 49 nt long. When the upstream crosslinker was 24 nt from the 3" end of the RNA (29-nt RNA), alpha was also contacted in the absence of NusA. The addition of NusA prevented RNA crosslinking to alpha. When the crosslinker was 44 nt from the 3' end (49-nt RNA), alpha crosslinks were still observed, but crosslinks to beta or beta' and NusA were greatly diminished. RNA crosslinking to alpha, and loss of this crosslink when NusA was added, was observed in the presence of NusB, NusE, and NusG and when transcription was carried out in the presence of an E. coli S100 cell extract. Peptide mapping localized the RNA interactions to the C-terminal domain of alpha.

Loss of Apc heterozygosity and abnormal tissue building in nascent intestinal polyps in mice carrying a truncated Apc gene.

Mutations in the APC (adenomatous polyposis coli) gene appear to be responsible for not only familial adenomatous polyposis but also many sporadic cases of gastrointestinal cancers. Using homologous recombination in mouse embryonic stem cells, we constructed mice that contained a mutant gene encoding a product truncated at a 716 (Apc delta 716). Mendelian transmission of the gene caused most homozygous mice to die in utero before day 8 of gestation. The heterozygotes developed multiple polyps throughout the intestinal tract, mostly in the small intestine. The earliest polyps arose multifocally during the third week after birth, and new polyps continued to appear thereafter. Surprisingly, every nascent polyp consisted of a microadenoma covered with a layer of the normal villous epithelium. These microadenomas originated from single crypts by forming abnormal outpockets into the inner (lacteal) side of the neighboring villi. We carefully dissected such microadenomas from nascent polyps by peeling off the normal epithelium and determined their genotype by PCR: all microadenomas had already lost the wild-type Apc allele, whereas the mutant allele remained unchanged. These results indicate that loss of heterozygosity followed by formation of intravillous microadenomas is responsible for polyposis in Apc delta 716 intestinal mucosa. It is therefore unlikely that the truncated product interacts directly with the wild-type protein and causes the microadenomas by a dominant negative mechanism.

Folding of a nascent polypeptide chain in vitro: cooperative formation of structure in a protein module.

We have prepared a family of peptide fragments of the 64-residue chymotrypsin inhibitor 2, corresponding to its progressive elongation from the N terminus. The growing polypeptide chain has little tendency to form stable structure until it is largely synthesized, and what structures are formed are nonnative and lack, in particular, the native secondary structural elements of alpha-helix and beta-sheet. These elements then develop as sufficient tertiary interactions are made in the nearly full-length chain. The growth of structure in the small module is highly cooperative and does not result from the hierarchical accretion of substructures.

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Moving on from nascent entrepreneurship:measuring cross-national differences in the transition to new business ownership

Nascent entrepreneurship and new business ownership are subsequent stages in the entrepreneurial process. We illustrate how information from the largest internationally harmonized database on entrepreneurship, the Global Entrepreneurship Monitor project, can be used to approximate the entrepreneurial process. We make a methodological contribution by computing the ratio of new business ownership to nascent entrepreneurship in a way that reflects the transition from nascent to new business ownership and provides cross-nationally comparable information on the efficiency of the entrepreneurial process for 48 countries. We report evidence for the validity of the transition ratio by benchmarking it against transition rates obtained from longitudinal studies and by correlating it with commonly used entrepreneurship indicators and macro-level economic indices. The transition ratio enables future cross-national research on the entrepreneurial process by providing a reliable and valid indicator for one key transition in this process. © 2012 Springer Science+Business Media New York.

The influence of socio-cultural environments on the performance of nascent entrepreneurs:community culture, motivation, self-efficacy and start-up success

The importance of informal institutions and in particular culture for entrepreneurship is a subject of ongoing interest. Past research has mostly concentrated on cross-national comparisons, cultural values and the direct effects of culture on entrepreneurial behaviour, but in the main found inconsistent results. We add a fresh perspective to this research stream by turning attention to community-level culture and cultural norms. We hypothesize indirect effects of cultural norms on venture emergence: Community-level cultural norms (performance-based culture and socially supportive institutional norms) impact important supply-side variables (entrepreneurial self-efficacy and entrepreneurial motivation) which in turn influence nascent entrepreneurs' success in creating operational ventures (venture emergence). We test our predictions on a unique longitudinal dataset, tracking nascent entrepreneurs' venture creation efforts over a five-year time span, and find evidence supporting them. Our research contributes to a more fine-grained understanding of how culture, in particular perceptions of community cultural norms, influences venture emergence. Based on these findings, we discuss how venture creation efforts can be supported. Our research highlights the embeddedness of entrepreneurial behaviour and its immediate antecedent beliefs in the local, community context. © 2012 Copyright Taylor and Francis Group, LLC.

The influence of socio-cultural environments on the performance of nascent entrepreneurs:an instrumental variable approach

The importance of informal institutions and in particular culture for entrepreneurship is a subject of ongoing interest. Past research has mostly concentrated on cross-national comparisons, cultural values, and the direct effects of culture on entrepreneurial behavior, but in the main found inconsistent results. The present research adds a fresh perspective to this research stream by turning attention to community-level culture and cultural norms. We hypothesize indirect effects of cultural norms on venture emergence. Specifically that community-level cultural norms (performance-based culture and socially-supportive institutional norms) impact important supply-side variables (entrepreneurial self-efficacy and entrepreneurial motivation) which in turn influence nascent entrepreneurs’ success in creating operational ventures (venture emergence). We test our predictions on a unique longitudinal data set (PSED II) tracking nascent entrepreneurs venture creation efforts over a 5 year time span and find evidence supporting them. Our research contributes to a more fine-grained understanding of how culture, in particular perceptions of community cultural norms, influences venture emergence. This research highlights the embeddedness of entrepreneurial behavior and its immediate antecedent beliefs in the local, community context.