c-Myc regulates transcriptional pause release.

Recruitment of the RNA polymerase II (Pol II) transcription initiation apparatus to promoters by specific DNA-binding transcription factors is well recognized as a key regulatory step in gene expression. We report here that promoter-proximal pausing is a general feature of transcription by Pol II in mammalian cells and thus an additional step where regulation of gene expression occurs. This suggests that some transcription factors recruit the transcription apparatus to promoters, whereas others effect promoter-proximal pause release. Indeed, we find that the transcription factor c-Myc, a key regulator of cellular proliferation, plays a major role in Pol II pause release rather than Pol II recruitment at its target genes. We discuss the implications of these results for the role of c-Myc amplification in human cancer.

RNA sequence analysis defines Dicer's role in mouse embryonic stem cells.

Short RNA expression was analyzed from Dicer-positive and Dicer-knockout mouse embryonic [corrected] stem (ES) cells, using high-throughput pyrosequencing. A correlation of miRNA quantification with sequencing frequency estimates that there are 110,000 miRNAs per ES cell, the majority of which can be accounted for by six distinct miRNA loci. Four of these miRNA loci or their human homologues have demonstrated roles in cell cycle regulation or oncogenesis, suggesting that a major function of the miRNA pathway in ES cells may be to shape their distinct cell cycle. Forty-six previously uncharacterized miRNAs were identified, most of which are expressed at low levels and are less conserved than the set of known miRNAs. Low-abundance short RNAs matching all classes of repetitive elements were present in cells lacking Dicer, although the production of some SINE- and simple repeat-associated short RNAs appeared to be Dicer-dependent. These and other Dicer-dependent sequences resembled miRNAs. At a depth of sequencing that approaches the total number of 5' phosphorylated short RNAs per cell, miRNAs appeared to be Dicer's only substrate. The results presented suggest a model in which repeat-associated miRNAs serve as host defenses against repetitive elements, a function canonically ascribed to other classes of short RNA.

Divergent transcription: a new feature of active promoters.

Divergent transcription is common of many promoters in organisms as diverse as mammals and yeast. Many independent experiments indicate that RNA polymerase II is frequently initiated but paused in the sense direction downstream from the promoter. Similarly a second peak of transcriptionally-engaged polymerase is paused in the anti-sense direction upstream of the promoter. Chromatin modifications that are associated with active promoters reside in nucleosomes immediately flanking this pair of paused polymerases. The nucleosome-free region associated with most promoters could in part be defined by this divergent transcription. The potential implications of divergent transcription on gene regulation and possible mechanisms that give rise to this phenomenon are discussed.

Small RNAs tell big stories in Whistler.

The Keystone Symposium on RNAi, microRNA and non-coding RNA convened on March 25-30 at Whistler Resort in Whistler, British Columbia, Canada. Researchers with backgrounds in different biochemical disciplines came together to exchange ideas on short RNAs and their roles in a host of biological processes.

Divergent transcription from active promoters.

Transcription initiation by RNA polymerase II (RNAPII) is thought to occur unidirectionally from most genes. Here, we present evidence of widespread divergent transcription at protein-encoding gene promoters. Transcription start site-associated RNAs (TSSa-RNAs) nonrandomly flank active promoters, with peaks of antisense and sense short RNAs at 250 nucleotides upstream and 50 nucleotides downstream of TSSs, respectively. Northern analysis shows that TSSa-RNAs are subsets of an RNA population 20 to 90 nucleotides in length. Promoter-associated RNAPII and H3K4-trimethylated histones, transcription initiation hallmarks, colocalize at sense and antisense TSSa-RNA positions; however, H3K79-dimethylated histones, characteristic of elongating RNAPII, are only present downstream of TSSs. These results suggest that divergent transcription over short distances is common for active promoters and may help promoter regions maintain a state poised for subsequent regulation.

Uncovering the enzymatic pKa of the ribosomal peptidyl transferase reaction utilizing a fluorinated puromycin derivative.

The ribosome-catalyzed peptidyl transferase reaction displays a complex pH profile resulting from two functional groups whose deprotonation is important for the reaction, one within the A-site substrate and a second unidentified group thought to reside in the rRNA peptidyl transferase center. Here we report the synthesis and activity of the beta,beta-difluorophenylalanyl derivative of puromycin, an A-site substrate. The fluorine atoms reduce the pK(a) of the nucleophilic alpha-amino group (<5.0) such that it is deprotonated at all pHs amenable to ribosomal analysis (pH 5.2-9.5). In the 50S modified fragment assay, this substrate reacts substantially faster than puromycin at neutral or acidic pH. The reaction follows a simplified pH profile that is dependent only upon deprotonation of a titratable group within the ribosomal active site. This feature will simplify characterization of the peptidyl transferase reaction mechanism. On the basis of the reaction efficiency of the doubly fluorinated substrate compared to the unfluorinated derivative, the Bronsted coefficient for the nucleophile is estimated to be substantially smaller than that reported for uncatalyzed aminolysis reactions, which has important mechanistic implications for the peptidyl transferase reaction.

Kinetic isotope effect analysis of the ribosomal peptidyl transferase reaction.

The ribosome is the macromolecular machine responsible for protein synthesis in all cells. Here, we establish a kinetic framework for the 50S modified fragment reaction that makes it possible to measure the kinetic effects that result from isotopic substitution in either the A or P site of the ribosome. This simplified peptidyl transferase assay follows a rapid equilibrium random mechanism in which the reverse reaction is nonexistent and the forward commitment is negligible. A normal effect (1.009) is observed for (15)N substitution of the incoming nucleophile at both low and high pH. This suggests that the first irreversible step is the formation of the tetrahedral intermediate. The observation of a normal isotope effect that does not change as a function of pH suggests that the ribosome promotes peptide bond formation by a mechanism that differs in its details from an uncatalyzed aminolysis reaction in solution. This implies that the ribosome contributes chemically to catalysis of peptide bond formation.

Evidence against stabilization of the transition state oxyanion by a pKa-perturbed RNA base in the peptidyl transferase center.

The crystal structure of the ribosomal 50S subunit from Haloarcula marismortui in complex with the transition state analog CCdA-phosphate-puromycin (CCdApPmn) led to a mechanistic proposal wherein the universally conversed A2451 in the ribosomal active site acts as an "oxyanion hole" to promote the peptidyl transferase reaction [Nissen, P., Hansen, J., Ban, N., Moore, P.B., and Steitz, T.A. (2000) Science 289, 920-929]. In the model, close proximity (3 A) between the A2451 N3 and the nonbridging phosphoramidate oxygen of CCdApPmn suggested that the carbonyl oxyanion formed during the tetrahedral transition state is stabilized by hydrogen bonding to the protonated A2451 N3, the pKa of which must be perturbed substantially. We characterize the contribution of the putative hydrogen bond between the N3 of A2451 and the nonbridging phosphoramidate oxygen by using chemical protection and peptidyl transfer inhibition assays. If this putative hydrogen bond makes a significant thermodynamic contribution, then CCdApPmn-binding affinity to the 50S ribosomal subunit should be strongly pH-dependent, with affinity increasing as the pH is lowered. We report that CCdApPmn binds 50S ribosomes with essentially equal affinity at all pH values between 5.0 and 8.5. These data argue against a mechanism for peptidyl transfer in which a residue with near neutral pKa stabilizes the transition-state oxyanion, at least to the extent that CCdApPmn accurately mimics the transition state.

A pre-translocational intermediate in protein synthesis observed in crystals of enzymatically active 50S subunits.

The large ribosomal subunit catalyzes peptide bond formation during protein synthesis. Its peptidyl transferase activity has often been studied using a 'fragment assay' that depends on high concentrations of methanol or ethanol. Here we describe a version of this assay that does not require alcohol and use it to show, both crystallographically and biochemically, that crystals of the large ribosomal subunits from Haloarcula marismortui are enzymatically active. Addition of these crystals to solutions containing substrates results in formation of products, which ceases when crystals are removed. When substrates are diffused into large subunit crystals, the subsequent structure shows that products have formed. The CC-puromycin-peptide product is found bound to the A-site and the deacylated CCA is bound to the P-site, with its 3prime prime or minute OH near N3 A2486 (Escherichia coli A2451). Thus, this structure represents a state that occurs after peptide bond formation but before the hybrid state of protein synthesis.