The organization of DNA into chromatin is thought to regulate gene expression in eukaryotes. To study its structure in vitro, there is a need for techniques that can isolate specific chromosomal loci of natively assembled chromatin. Current purification methods often involve chemical cross-linking to preserve the chromatin composition. However, such cross-linking may affect the native structure. It also impedes single molecule force spectroscopy experiments, which have been instrumental to probe chromatin folding. Here we present a method for the incorporation of affinity tags, such as biotin, into native nucleoprotein fragments based on their DNA sequence, and subsequent single molecule analysis by magnetic tweezers. DNA oligos with several Locked Nucleic Acid (LNA) nucleotides are shown to selectively bind to target DNA at room temperature, mediated by a toehold end in the target, allowing for selective purification of DNA fragments. The stability of the probe-target hybrid is sufficient to withstand over 65 pN of force. We employ these probes to obtain force-extension curves of native chromatin fragments of the 18S ribosomal DNA from the yeast Saccharomyces cerevisiae. These experiments yield valuable insights in the heterogeneity in structure and composition of natively assembled chromatin at the single-molecule level.

RNA polymerase I (Pol I) synthesizes ribosomal RNA (rRNA) in all eukaryotes, accounting for the major part of transcriptional activity in proliferating cells. Although basal Pol I transcription factors have been characterized in diverse organisms, the molecular basis of the robust rRNA production in vivo remains largely unknown. In S. cerevisiae, the multifunctional Net1 protein was reported to stimulate Pol I transcription. We found that the Pol I-stimulating function can be attributed to the very C-terminal region (CTR) of Net1. The CTR was required for normal cell growth and Pol I recruitment to rRNA genes in vivo and sufficient to promote Pol I transcription in vitro. Similarity with the acidic tail region of mammalian Pol I transcription factor UBF, which could partly functionally substitute for the CTR, suggests conserved roles for CTR-like domains in Pol I transcription from yeast to human.

In growing eukaryotic cells, nuclear ribosomal (r)RNA synthesis by RNA polymerase (RNAP) I accounts for the vast majority of cellular transcription. This high output is achieved by the presence of multiple copies of rRNA genes in eukaryotic genomes transcribed at a high rate. In contrast to most of the other transcribed genomic loci, actively transcribed rRNA genes are largely devoid of nucleosomes adapting a characteristic “open” chromatin state, whereas a significant fraction of rRNA genes resides in a transcriptionally inactive nucleosomal “closed” chromatin state. Here, we review our current knowledge about the nature of open rRNA gene chromatin and discuss how this state may be established.

Nuclear eukaryotic RNA polymerases (RNAPs) transcribe a chromatin template in vivo. Since the basic unit of chromatin, the nucleosome, renders the DNA largely inaccessible, RNAPs have to overcome the nucleosomal barrier for efficient RNA synthesis. Gaining mechanistical insights in the transcription of chromatin templates will be essential to understand the complex process of eukaryotic gene expression. In this article we describe the use of defined in vitro transcription systems for comparative analysis of highly purified RNAPs I–III from S. cerevisiae (hereafter called yeast) transcribing in vitro reconstituted nucleosomal templates. We also provide a protocol to study promoter-dependent RNAP I transcription of purified native 35S ribosomal RNA (rRNA) gene chromatin.

In archaea and bacteria the major classes of RNAs are synthesized by one DNA-dependent RNA polymerase (RNAP). In contrast, most eukaryotes have three highly specialized RNAPs to transcribe the nuclear genome. RNAP I synthesizes almost exclusively ribosomal (r)RNA, RNAP II synthesizes mRNA as well as many noncoding RNAs involved in RNA processing or RNA silencing pathways and RNAP III synthesizes mainly tRNA and 5S rRNA. This review discusses functional differences of the three nuclear core RNAPs in the yeast S. cerevisiae with a particular focus on RNAP I transcription of nucleolar ribosomal (r)DNA chromatin.