Serine-7 of the RNA polymerase II CTD is specifically required for snRNA gene expression

RNA polymerase II (Pol II) transcribes genes that encode proteins and noncoding small nuclear RNAs (snRNAs). The carboxyl-terminal repeat domain (CTD) of the largest subunit of mammalian RNA Pol II, comprising tandem repeats of the heptapeptide consensus Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7, is required for expression of both gene types. We show that mutation of serine-7 to alanine causes a specific defect in snRNA gene expression. We also present evidence that phosphorylation of serine-7 facilitates interaction with the snRNA gene-specific Integrator complex. These findings assign a biological function to this amino acid and highlight a gene type-specific requirement for a residue within the CTD heptapeptide, supporting the existence of a CTD code.     

A role for interaction of the RNA polymerase flap domain with the sigma subunit in promoter recognition

In bacteria, promoter recognition depends on the RNA polymerase sigma subunit, which combines with the catalytically proficient RNA polymerase core to form the holoenzyme. The major class of bacterial promoters is defined by two conserved elements (the -10 and -35 elements, which are 10 and 35 nucleotides upstream of the initiation point, respectively) that are contacted by sigma in the holoenzyme. We show that recognition of promoters of this class depends on the "flexible flap" domain of the RNA polymerase beta subunit. The flap interacts with conserved region 4 of sigma and triggers a conformational change that moves region 4 into the correct position for interaction with the -35 element. Because the flexible flap is evolutionarily conserved, this domain may facilitate promoter recognition by specificity factors in eukaryotes as well.     

Structural roles for human translation factor eIF3 in initiation of protein synthesis

Protein synthesis in mammalian cells requires initiation factor eIF3, a approximately 750-kilodalton complex that controls assembly of 40S ribosomal subunits on messenger RNAs (mRNAs) bearing either a 5'-cap or an internal ribosome entry site (IRES). Cryo-electron microscopy reconstructions show that eIF3, a five-lobed particle, interacts with the hepatitis C virus (HCV) IRES RNA and the 5'-cap binding complex eIF4F via the same domain. Detailed modeling of eIF3 and eIF4F onto the 40S ribosomal subunit reveals that eIF3 uses eIF4F or the HCV IRES in structurally similar ways to position the mRNA strand near the exit site of 40S, promoting initiation complex assembly.     

Recognition of histone H3 lysine-4 methylation by the double tudor domain of JMJD2A

Biological responses to histone methylation critically depend on the faithful readout and transduction of the methyl-lysine signal by "effector" proteins, yet our understanding of methyl-lysine recognition has so far been limited to the study of histone binding by chromodomain and WD40-repeat proteins. The double tudor domain of JMJD2A, a Jmjc domain-containing histone demethylase, binds methylated histone H3-K4 and H4-K20. We found that the double tudor domain has an interdigitated structure, and the unusual fold is required for its ability to bind methylated histone tails. The cocrystal structure of the JMJD2A double tudor domain with a trimethylated H3-K4 peptide reveals that the trimethyl-K4 is bound in a cage of three aromatic residues, two of which are from the tudor-2 motif, whereas the binding specificity is determined by side-chain interactions involving amino acids from the tudor-1 motif. Our study provides mechanistic insights into recognition of methylated histone tails by tudor domains and reveals the structural intricacy of methyl-lysine recognition by two closely spaced effector domains.     

Regulation of replication fork progression through histone supply and demand

DNA replication in eukaryotes requires nucleosome disruption ahead of the replication fork and reassembly behind. An unresolved issue concerns how histone dynamics are coordinated with fork progression to maintain chromosomal stability. Here, we characterize a complex in which the human histone chaperone Asf1 and MCM2-7, the putative replicative helicase, are connected through a histone H3-H4 bridge. Depletion of Asf1 by RNA interference impedes DNA unwinding at replication sites, and similar defects arise from overproduction of new histone H3-H4 that compromises Asf1 function. These data link Asf1 chaperone function, histone supply, and replicative unwinding of DNA in chromatin. We propose that Asf1, as a histone acceptor and donor, handles parental and new histones at the replication fork via an Asf1-(H3-H4)-MCM2-7 intermediate and thus provides a means to fine-tune replication fork progression and histone supply and demand.     

Crystal structure of an initiation factor bound to the 30S ribosomal subunit

Initiation of translation at the correct position on messenger RNA is essential for accurate protein synthesis. In prokaryotes, this process requires three initiation factors: IF1, IF2, and IF3. Here we report the crystal structure of a complex of IF1 and the 30S ribosomal subunit. Binding of IF1 occludes the ribosomal A site and flips out the functionally important bases A1492 and A1493 from helix 44 of 16S RNA, burying them in pockets in IF1. The binding of IF1 causes long-range changes in the conformation of H44 and leads to movement of the domains of 30S with respect to each other. The structure explains how localized changes at the ribosomal A site lead to global alterations in the conformation of the 30S subunit.     

Structure of the S15,S6,S18-rRNA complex: assembly of the 30S ribosome central domain

The crystal structure of a 70-kilodalton ribonucleoprotein complex from the central domain of the Thermus thermophilus 30S ribosomal subunit was solved at 2.6 angstrom resolution. The complex consists of a 104-nucleotide RNA fragment composed of two three-helix junctions that lie at the end of a central helix, and the ribosomal proteins S15, S6, and S18. S15 binds the ribosomal RNA early in the assembly of the 30S ribosomal subunit, stabilizing a conformational reorganization of the two three-helix junctions that creates the RNA fold necessary for subsequent binding of S6 and S18. The structure of the complex demonstrates the central role of S15-induced reorganization of central domain RNA for the subsequent steps of ribosome assembly.     

Function and molecular mechanism of acetylation in autophagy regulation

Protein acetylation emerged as a key regulatory mechanism for many cellular processes. We used genetic analysis of Saccharomyces cerevisiae to identify Esa1 as a histone acetyltransferase required for autophagy. We further identified the autophagy signaling component Atg3 as a substrate for Esa1. Specifically, acetylation of K19 and K48 of Atg3 regulated autophagy by controlling Atg3 and Atg8 interaction and lipidation of Atg8. Starvation induced transient K19-K48 acetylation through spatial and temporal regulation of the localization of acetylase Esa1 and the deacetylase Rpd3 on pre-autophagosomal structures (PASs) and their interaction with Atg3. Attenuation of K19-K48 acetylation was associated with attenuation of autophagy. Increased K19-K48 acetylation after deletion of the deacetylase Rpd3 caused increased autophagy. Thus, protein acetylation contributes to control of autophagy.