Shaken, not stirred

The pathology of several neurodegenerative diseases involves the accumulation of ordered protein aggregates called amyloid fibrils. These self-assembled filaments are characterized by a high β-sheet content with the strands organized perpendicular to the fibril axis. Despite the common structural organization, amyloid fibrils often have different morphologies when viewed under either electron or atomic force microscopes. To understand the origin of these distinct morphologies at the molecular level, Tycko and colleagues studied the fibrils formed by a 40-residue peptide associated with Alzheimer disease (Aβ1–40). They showed that subtle differences in the incubation conditions, such as no stirring or gentle shaking, could alter the types of Aβ1–40 fibrils that predominate in the sample. In addition, once formed, these morphologically different fibrils self-propagate independently of the incubation conditions. Using solid-state NMR the authors show that, within the different types of fibrils, several amino acid residues of the Aβ1–40 peptide are in different local structures and/or conformational environments. Notably, although the fibrils obtained under the two incubation conditions are toxic to the cells, the type formed with no stirring is substantially more so than the other. These observations provide a glimpse of the molecular interactions present within fibrils of different morphologies and support the hypothesis that the phenomenon of 'strains' in prion disease may arise from similar structural variations in prion protein aggregates. (Science 307, 262–265, 2005) HPF

Bcl-2 mismatches

Bcl-2 is overexpressed in many types of cancer and is generally believed to promote carcinogenesis by inhibiting apoptosis. However, Bcl-2-mediated oncogenesis has also been attributed to increased rates of DNA mutagenesis. While it is known that Bcl-2 expression is able to stimulate mutagenesis after exposure to DNA-damaging agents, little is known about its mechanism of action. You and colleagues have examined the effects of Bcl-2 in normal human fibroblast and human B-cell lymphoma cell lines. They found that Bcl-2 may suppress mismatch repair (MMR) by acting indirectly on the transcription factor E2F and directly on cyclin dependent kinase 2 (Cdk2), independent of its anti-apoptotic activity. In cells expressing Bcl-2 at physiologically relevant levels, they found decreased levels of the essential MMR protein hMSH2, which is encoded by an E2F-responsive gene. In addition, they saw that Bcl-2 expression promoted the formation of an E2F–retinoblastoma protein (pRb) complex through hypophosphorylation of pRb. The formation of this complex prevents E2F from binding DNA, resulting in down-regulation of hMSH2 expression. Additional data suggested that pRb hypophosphorylation may be caused by the increased levels of Cdk inhibitors, like p27Kip1, triggered by Bcl-2 expression, as well as by the direct binding of Cdk2 by Bcl-2. Both processes inhibit phosphorylation of pRb by Cdk2 and lead to cell-cycle arrest. The authors suggest that Bcl-2-mediated cell-cycle arrest induces genetic instability by suppressing MMR, contributing to tumorigenesis. (Nat. Cell Biol. 7, 137–147, 2005) MM

The ribosome unwinds

Many mRNAs adopt complex secondary structures that the translation machinery must unfold because the mRNA codons must be in single-stranded form to get translated. Structural analyses suggested that disruption of the secondary structure of translating mRNA must occur either before the mRNA encounters or as it enters the 30S subunit of the ribosome. The identity of the enzyme that unwinds the mRNA helical structures has until now remained a mystery. This is in part due to the lack of a defined in vitro translation system in which the activity of the individual components of the translation machinery could be examined. In a recent paper, Noller and colleagues describe an in vitro system containing Escherichia coli ribosomes, mRNA, tRNAs and elongation factors EF-Tu and EF-G. This system is used with an RNA oligonucleotide displacement assay to monitor stepwise translocation, one codon at a time, and shows that only the ribosome has mRNA helicase activity. They localized the unwinding activity to the middle of the mRNA entry tunnel in the 30S subunit. Mutational studies indicated that the S3, S4 and S5 proteins, which line this entry tunnel and encircle the incoming mRNA, are important for the processivity of the ribosomal helicase. Noller and colleagues suggest that these ribosomal proteins may function in a similar manner to the sliding clamp, encircling the nucleic acid and preventing backsliding or dissociation of the RNA from the ribosome. However, the S3, S4 and S5 proteins do not contain canonical helicase domains and the helicase activity of the ribosome does not require ATP, suggesting that the ribosome uses a novel type of helicase to unwind the mRNA. (Cell 120, 49–58, 2005) EJ

Into the jaws of RNA polymerase

Into the jaws of RNA polymerase The first steps in gene expression require DNA strand unpairing and placement of the transcription start site (+1) in the active site of RNA polymerase (RNAP). This process occurs through a series of distinct steps, each providing a point for regulatory input. Amounts of mRNA are often increased by the binding of activator proteins within and/or directly upstream of a promoter; these interactions enhance the stability of the initial RNA polymerase–promoter complex and/or the rates of subsequent isomerization steps that create the transcription bubble. While activators bind to specific DNA sequences, growing evidence (at least in prokaryotes) suggests that interactions of polymerase (specifically, the alpha subunit C-terminal domain) with upstream DNA by itself can influence the conformational changes required to form the open complex. Recent studies of the association of Escherichia coli RNAP with the λPR promoter (Saecker and colleagues) and the lac UV5 promoter (Ross and Gourse) find that the presence of DNA upstream of base pair -45 greatly accelerates the rate limiting step (isomerization) in open complex formation, a step which occurs after polymerase binding. High resolution structures of multisubunit RNAP place the active site at the bottom of a deep cleft; their conserved architecture dictates that downstream DNA bend upon entering the cleft. Using DNase I footprinting and other data, Saecker and colleagues infer that upstream contacts act early in initiation to facilitate entry of downstream DNA into the active site channel. They hypothesize that early wrapping interactions with upstream DNA directly influence RNAP conformation, facilitating DNA distortions in such a way that it can fully enter the jaws of RNA polymerase.(Proc. Natl. Acad. Sci. USA 102 285–290 and 291–296, 2005) BK