Nucleoprotein structure of adenovirus

The structure of the nucleoprotein core of adenovirus has been investigated. Using a combination of biophysical techniques and nuclease digestion I have determined that the adenovirus core, as isolated with pyridine, shares several physical properties with cellular chromatin. The most notable difference is the lack of a nucleosome repeat in the staph nuclease digestion intermediates from adenovirus cores. I have exploited this difference to study the fate of the nucleoprotein structure of adenovirus DNA early during productive infection. When nuclei from cells infected with ³²P-labeled adenovirus are digested with staph nuclease the pattern of labeled digestion intermediates more closely resembles the pattern of cellular intermediates. This result indicates that virus core proteins are replaced, early during infection, with cellular histones. The newly assembled adenovirus nucleosomes differ from the bulk of the cellular chromatin by having a shorter repeat (160 vs. 185 base pairs) and more labile linker and core regions. Experiments with inhibitors of cellular DNA synthesis indicate that adenovirus nucleosomes are assembled independently of cellular. DNA synthesis. Using the Southern blotting technique it is shown that newly synthesized adenovirus DNA is not in a nucleosome repeat late in infection. At this time of infection, however, the input adenovirus genomes persist in a nucleosome structure. The results of these experiments are discussed in terms of the template requirements for eucaryotic transcription and replication

Transcriptome-Wide Binding Sites for Components of the Saccharomyces cerevisiae Non-Poly(A) Termination Pathway: Nrd1, Nab3, and Sen1

RNA polymerase II synthesizes a diverse set of transcripts including both protein-coding and non-coding RNAs. One major difference between these two classes of transcripts is the mechanism of termination. Messenger RNA transcripts terminate downstream of the coding region in a process that is coupled to cleavage and polyadenylation reactions. Non-coding transcripts like Saccharomyces cerevisiae snoRNAs terminate in a process that requires the RNA–binding proteins Nrd1, Nab3, and Sen1. We report here the transcriptome-wide distribution of these termination factors. These data sets derived from in vivo protein–RNA cross-linking provide high-resolution definition of non-poly(A) terminators, identify novel genes regulated by attenuation of nascent transcripts close to the promoter, and demonstrate the widespread occurrence of Nrd1-bound 3′ antisense transcripts on genes that are poorly expressed. In addition, we show that Sen1 does not cross-link efficiently to many expected non-coding RNAs but does cross-link to the 3′ end of most pre–mRNA transcripts, suggesting an extensive role in mRNA 3′ end formation and/or termination

Genome-Wide Mapping of Yeast RNA Polymerase II Termination

<div><p>Yeast RNA polymerase II (Pol II) terminates transcription of coding transcripts through the polyadenylation (pA) pathway and non-coding transcripts through the non-polyadenylation (non-pA) pathway. We have used PAR-CLIP to map the position of Pol II genome-wide in living yeast cells after depletion of components of either the pA or non-pA termination complexes. We show here that Ysh1, responsible for cleavage at the pA site, is required for efficient removal of Pol II from the template. Depletion of Ysh1 from the nucleus does not, however, lead to readthrough transcription. In contrast, depletion of the termination factor Nrd1 leads to widespread runaway elongation of non-pA transcripts. Depletion of Sen1 also leads to readthrough at non-pA terminators, but in contrast to Nrd1, this readthrough is less processive, or more susceptible to pausing. The data presented here provide delineation of <i>in vivo</i> Pol II termination regions and highlight differences in the sequences that signal termination of different classes of non-pA transcripts.</p></div

Ysh1 depletion causes readthrough at pA sites.

<p><b>A.</b> Mean reads every 10 bp for the 500 most frequently used pA sites with (dotted) and without (solid) Ysh1. <b>B.</b> Plot showing percent readthrough of each of 500 pA terminators as a fraction of the total 500 pA terminators. <b>C and D.</b> Histograms representing normalized reads with Ysh1 (grey) and without Ysh1 (black) at the given genomic locations. The TSS with the direction of transcription is indicated by an arrow. Genes and pA sites are represented below each graph and the length of the genome depicted is given in the lower right hand corner.</p

Interaction of yeast RNA-binding proteins Nrd1 and Nab3 with RNA polymerase II terminator elements

Yeast RNA-binding proteins Nrd1 and Nab3 direct transcription termination of sn/snoRNA transcripts, some mRNA transcripts, and a class of intergenic and anti-sense transcripts. Recognition of Nrd1- and Nab3-binding sites is a critical first step in the termination and subsequent processing or degradation of these transcripts. In this article, we describe the purification and characterization of an Nrd1–Nab3 heterodimer. This Nrd1–Nab3 complex binds specifically to RNA sequences derived from a snoRNA terminator. The relative binding to mutant terminators correlates with the in vivo termination efficiency of these mutations, indicating that the primary specificity determinant in nonpoly(A) termination is Nrd1–Nab3 binding. In addition, several snoRNA terminators contain multiple Nrd1- and Nab3-binding sites and we show that multiple heterodimers bind cooperatively to one of these terminators in vitro

Schematic representation of Pol II termination after removal of non-pA and pA termination factors.

<p>Elongating Pol II (green) terminates pA transcripts (A) after an allosteric change (red) that reduces processivity. (B) Depletion of Ysh1 leads to minimally extended readthrough transcripts but does not block the allosteric change in Pol II. (C) Nrd1 and Nab3 binding recruit Sen1 for termination of non-pA transcripts. (D) Pol II elongation complex lacking Nrd1 does not recognize termination sequences in the nascent transcript and thus does not facilitate the allosteric transition in Pol II. This leads to processive readthrough. (E) Nrd1 and Nab3 recognize terminator sequences allowing the allosteric change in Pol II but depletion of Sen1 blocks removal of Pol II from the template.</p