with the analysis of folding of specific endogenous proteins in mutant strains, these studies have led to signifi-Hermann Herder Str. 7 D-79104 Freiburg cant advances in our understanding of protein folding in the complex cellular milieu. Germany † Department of Biomolecular Chemistry The Welcoming Committee for Nascent Polypeptide Chains University of Wisconsin 1300 University Avenue Nascent chains emerging at the peptide exit tunnel of Madison, Wisconsin 53706 the ribosome are awaited by a welcoming committee of ribosome-associated chaperones. These factors have been most thoroughly studied in E. coli. Trigger factor (TF), which is found in all eubacteria analyzed, is the Cellular synthesis of polypeptides is an amazing, commajor protein that cross-links to virtually all nascent plex, and efficient process. An E. coli cell utilizes up to chains of secretory and cytosolic proteins tested (Valent 20,000 ribosomes to produce an estimated total of et al., 1995; Hesterkamp et al., 1996). It may be posi-30,000 polypeptides per minute. Yet it is only the begintioned close to the exit site, as it cross-links to ribosomening. Each newly made protein must be folded into its associated chains 57 residues in length. TF, which has a correct tertiary structure. How this is achieved is one of central domain with homology to FK506 binding proteins the most basic, and complicated, questions of molecular (FKBPs), displays peptidyl-prolyl-cis-trans isomerase biology. and chaperone-like activities in vitro. It is unclear which When does the process of folding begin in the lifetime of these activities is required for its function in vivo. of a protein? Crystallographic data of bacterial ribo-Recent genetic studies provide clues suggesting a somes identified a peptide exit tunnel in the large subunit role of TF in protein folding in vivo. ⌬tig mutants lacking with a length of 100 Å and an average diameter of about TF have no apparent phenotype and no major defects 20 Å (Ban et al., 1999). This tunnel is long enough to in folding of newly synthesized proteins (Deuerling et accommodate extended chains of about 30 residues al., 1999; Teter et al., 1999). However, in the absence of and perhaps longer peptides in helical conformation, the Hsp70 chaperone DnaK, ⌬tig mutants are inviable. but certainly not peptides with tertiary structure. Once Upon depletion of DnaK in ⌬tig strains, more than 40 the N-terminal residues are synthesized, the nascent different newly synthesized cytosolic proteins aggrepolypeptide emerges into the crowded environment of gate, indicating a role for TF in folding of cytosolic prothe cytosol. It is well established that nascent proteins teins in cooperation with the DnaK chaperone system can fold cotranslationally (i.e., while still attached to (see below). ribosomes) in cell-free translation systems. Recently, TF binds in vitro with 1:1 stoichiometry to the large a study using the Semliki Forest virus capsid protein subunits of both translating and nontranslating riboestablished that cotranslational folding can...

Purpose To determine whether a structured mentoring curriculum improves research mentoring skills. Method The authors conducted a randomized controlled trial (RCT) at 16 academic health centers (June 2010 to July 2011). Faculty mentors of trainees who were conducting clinical/translational research ≥50% of the time were eligible. The intervention was an eight-hour, case-based curriculum focused on six mentoring competencies. The primary outcome was the change in mentors’ self-reported pretest to posttest composite scores on the Mentoring Competency Assessment (MCA). Secondary outcomes included changes in the following: mentors’ awareness as measured by their self-reported retrospective change in MCA scores, mentees’ ratings of their mentors’ competency as measured by MCA scores, and mentoring behaviors as reported by mentors and their mentees. Results A total of 283 mentor–mentee pairs were enrolled: 144 mentors were randomized to the intervention; 139 to the control condition. Self-reported pre-/posttest change in MCA composite scores was higher for mentors in the intervention group compared with controls (P < .001). Retrospective changes in MCA composite scores between the two groups were even greater, and extended to all six subscale scores (P < .001). More intervention-group mentors reported changes in their mentoring practices than control mentors (P < .001). Mentees working with intervention-group mentors reported larger changes in retrospective MCA pre-/posttest scores (P = .003) and more changes in their mentors’ behavior (P = .002) than those paired with control mentors. Conclusions This RCT demonstrates that a competency-based research mentor training program can improve mentors’ skills.

The 70 kDa heat shock proteins (Hsp70s) are a ubiquitous class of molecular chaperones. The Ssbs of Saccharomyces cerevisiae are an abundant type of Hsp70 found associated with translating ribosomes. To understand better the function of Ssb in association with ribosomes, the Ssb-ribosome interaction was characterized. Incorporation of the aminoacyl-tRNA analog puromycin by translating ribosomes caused the release of Ssb concomitant with the release of nascent chains. In addition, Ssb could be cross-linked to nascent chains containing a modified lysine residue with a photoactivatable cross-linker. Together, these results suggest an interaction of Ssb with the nascent chain. The interaction of Ssb with the ribosome-nascent chain complex was stable, as demonstrated by resistance to treatment with high salt; however, Ssb interaction with the ribosome in the absence of nascent chain was salt sensitive. We propose that Ssb is a core component of the translating ribosome which interacts with both the nascent polypeptide chain and the ribosome. These interactions allow Ssb to function as a chaperone on the ribosome, preventing the misfolding of newly synthesized proteins.

Bacterial flagellins have been portrayed as a relatively invariant pathogen-associated molecular pattern. We have found within-species, within-pathovar variation for defense-eliciting activity of flagellins among Xanthomonas campestris pv campestris (Xcc) strains. Arabidopsis thaliana FLAGELLIN SENSING2 (FLS2), a transmembrane leucine-rich repeat kinase, confers flagellin responsiveness. The flg22 region was the only Xcc flagellin region responsible for detectable elicitation of Arabidopsis defense responses. A Val-43/Asp polymorphism determined the eliciting/noneliciting nature of Xcc flagellins (structural gene fliC). Arabidopsis detected flagellins carrying Asp-43 or Asn-43 but not Val-43 or Ala-43, and it responded minimally for Glu-43. Wild-type Xcc strains carrying nonrecognized flagellin were more virulent than those carrying a recognized flagellin when infiltrated into Arabidopsis leaf mesophyll, but this correlation was misleading. Isogenic Xcc fliC gene replacement strains expressing eliciting or noneliciting flagellins grew similarly, both in leaf mesophyll and in hydathode/vascular colonization assays. The plant FLS2 genotype also had no detectable effect on disease outcome when previously untreated plants were infected by Xcc. However, resistance against Xcc was enhanced if FLS2-dependent responses were elicited 1 d before Xcc infection. Prior immunization was not required for FLS2-dependent restriction of Pseudomonas syringae pv tomato. We conclude that plant immune systems do not uniformly detect all flagellins of a particular pathogen species and that Xcc can evade Arabidopsis FLS2-mediated defenses unless the FLS2 system has been activated by previous infections.

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