Bacteria use multiple sigma factors to coordinate gene expression in response to environmental perturbations. In Escherichia coli and other γ-proteobacteria, the transcription factor Crl stimulates σ Sdependent transcription during times of cellular stress by promoting the association of σ S with core RNA polymerase. The molecular basis for specific recognition of σ S by Crl, rather than the homologous and more abundant primary sigma factor σ 70 , is unknown. Here we use bacterial two-hybrid analysis in vivo and p-benzoylphenylalanine cross-linking in vitro to define the features in σ S responsible for specific recognition by Crl. We identify residues in σ S conserved domain 2 (σ S 2 ) that are necessary and sufficient to allow recognition of σ 70 conserved domain 2 by Crl, one near the promoter-melting region and the other at the position where a large nonconserved region interrupts the sequence of σ 70 . We then use luminescence resonance energy transfer to demonstrate directly that Crl promotes holoenzyme assembly using these specificity determinants on σ S . Our results explain how Crl distinguishes between sigma factors that are largely homologous and activates discrete sets of promoters even though it does not bind to promoter DNA.RNAP formation | transcription initiation | bacterial stress response | RpoS | curli fiber T ranscription initiation in bacteria requires the assembly of a sigma factor (σ) with the RNA polymerase (RNAP) catalytic core (E, composed of 2 α-subunits and one each of β, β′, and ω) to form RNAP holoenzyme (Eσ), which in turn recognizes promoter sequences (1) (reviewed in ref. 2). Multiple sigma factors compete for binding to core RNAP (reviewed in refs. 3,4), and each sigma factor controls a specific set of promoters.In Escherichia coli, which has seven sigma factors, σ 70 is the primary sigma, and σ S is important for certain stress responses and during the stationary phase of growth (5). Eσ S -dependent transcription initiation is regulated by σ S , whose concentration is itself regulated at the levels of transcription, translation, and protein stability (reviewed in ref. 6). Eσ S -dependent transcription is also activated by Crl (7), a small protein that increases expression of many stress response genes and those required for formation of amyloid curli fibers (which accounts for its name) involved in adhesion and biofilm formation (reviewed in refs. 2,6,8).The effect of Crl on σ S -dependent transcription in vivo is most pronounced during the transition into stationary phase (9). It has been proposed that Crl functions by increasing the concentration of Eσ S holoenzyme by facilitating assembly of σ S with core RNAP (10-12) because Crl's effects on transcription are greatest in vitro when the concentration of σ S is lowest, and overexpression of σ S complements a crl deletion in vivo (13). Effects of Crl have also been reported on postholoenzyme assembly steps including promoter binding (14) and open complex formation (12).Sigma factors contain several protease-resistant domains, e...

Human cytomegalovirus (HCMV) is a pervasive and significant pathogen. At present, there is no HCMV vaccine, and the available drugs target only replication events. Thus, new therapeutic strategies are needed. HCMV fusion appears to require interactions of ␣-helical regions in viral surface glycoproteins gB and gH. Oligomers of ␤-amino acids ("␤-peptides") are attractive unnatural scaffolds for mimicry of specific protein surfaces, because ␤-peptides adopt predictable helical conformations and resist proteolysis. Here, we report the development of ␤-peptides designed to mimic the gB heptad repeat and block HCMV entry. The most potent ␤-peptide inhibits HCMV infection in a cell based-assay with an IC 50 of ϳ30 M. Consistent with our structure-based design strategy, inhibition is highly specific for HCMV relative to other related viruses. Mechanistic studies indicate that inhibitory ␤-peptides act by disrupting membrane fusion. Our findings raise the possibility that ␤-peptides may provide a general platform for development of a new class of antiviral agents and that inhibitory ␤-peptides will constitute new tools for elucidating viral entry mechanisms.

Tg2576 mice produce high levels of beta-amyloid (Abeta) and develop amyloid deposits, but lack neurofibrillary tangles and do not suffer the extensive neuronal cell loss characteristic of Alzheimer's disease. Protection from Abeta toxicity has been attributed to up-regulation of transthyretin (TTR), a normal component of plasma and cerebrospinal fluid. We compared the effect of TTR purified from human plasma (pTTR) with that produced recombinantly (rTTR) on Abeta aggregation and toxicity. pTTR slowed Abeta aggregation but failed to protect primary cortical neurons from Abeta toxicity. In contrast, rTTR accelerated aggregation, while effectively protecting neurons. This inverse correlation between Abeta aggregation kinetics and toxicity is consistent with the hypothesis that soluble intermediates rather than insoluble fibrils are the most toxic Abeta species. We carried out a detailed comparison of pTTR with rTTR to ascertain the probable cause of these different effects. No differences in secondary, tertiary or quaternary structure were detected. However, pTTR differed from rTTR in the extent and nature of modification at Cys10. We hypothesize that differential modification at Cys10 regulates TTR's effect on Abeta aggregation and toxicity.

Coactivator-associated arginine methyl transferase 1 (CARM1) is a protein arginine methyltransferase (PRMT) family member that functions as a coactivator in androgen and estrogen signaling pathways and plays a role in the progression of prostate and breast cancer. CARM1 catalyzes methylation of diverse protein substrates. Prior attempts to purify the full-length mouse CARM1 protein have proven unsatisfactory. The full-length protein expressed in E. coli forms insoluble inclusion bodies that are difficult to denature and to refold. The presented results demonstrate the use of a novel HaloTag™ technology to purify full-length CARM1 from both E. coli and mammalian HEK293T cells. A small amount of CARM1 was purified from E. coli; however, the protein was truncated on the N-terminus by 10–50 amino acids, most likely due to endogenous proteolytic activity. In contrast, substantial quantities of soluble full-length CARM1 were purified from transiently transfected HEK293T cells. The CARM1 from HEK293T cells was isolated alongside a number of co-purifying interacting proteins. The covalent bond formed between the HaloTag and the HaloLink resin allowed the use of stringent wash conditions without risk of eluting the CARM1 protein. The results also illustrate a highly effective approach for purifying and enriching both CARM1-associated proteins as well as substrates for CARM1’s methyltransferase activity.

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