Molecular Mechanisms of Transcription through Single-Molecule Experiments

Transcription represents the first step in gene expression. It is therefore not surprising that transcription is a highly regulated process and its control is essential to understand the flow and processing of information required by the cell to maintain its homeostasis. During transcription, a DNA molecule is copied into RNA molecules that are then used to translate the genetic information into proteins; this logical pattern has been conserved throughout all three kingdoms of life, from Archaea to Eukarya, making it an essential and fundamental cellular process. Even though some viruses that encode their genome in an RNA molecule use it as a template to make mRNA, others synthesize an intermediate DNA molecule from the RNA, a process known as reverse transcription, from which regular transcription of viral genes can then proceed in the host cells

Dissecting the Nucleosomal Barrier to Transcription

The nucleosome, which represents the fundamental unit of chromatin and an intrinsic regulator of transcription, consists of a histone octamer and ∼150 base pairs of DNA. The histone proteins in turn contain two functional regions: the histone-fold domains, which make strong contacts with the DNA and organize it into the superhelical structure specific to the nucleosome, and the histone tails, which are highly positively charged and can stabilize the nucleosome further. Both histone regions, but especially the tails, are the target of many posttranslational modifications associated with gene expression. © 2011 Biophysical Society. Published by Elsevier Inc

A Quantitative Kinetic Model of Eukaryotic Transcription Elongation from Single-Molecule Experiments

Transcription by RNA polymerase II (Pol II) is an important point of control for eukaryotic gene expression, and has been extensively studied by structural, biochemical, and biophysical methods. During transcription elongation, Pol II moves processively along the DNA template and synthesizes RNA, one nucleotide at a time. A comprehensive kinetic characterization of this process, the transcription elongation cycle, incorporating both the on-pathway nucleotide addition phase and the off-pathway pausing phase, is still lacking. © 2013 Biophysical Society. Published by Elsevier Inc

Complete dissection of transcription elongation reveals slow translocation of RNA polymerase II in a linear ratchet mechanism

During transcription elongation, RNA polymerase has been assumed to attain equilibrium between pre- and post-translocated states rapidly relative to the subsequent catalysis. Under this assumption, recent single-molecule studies proposed a branched Brownian ratchet mechanism that necessitates a putative secondary nucleotide binding site on the enzyme. By challenging individual yeast RNA polymerase II with a nucleosomal barrier, we separately measured the forward and reverse translocation rates. Surprisingly, we found that the forward translocation rate is comparable to the catalysis rate. This finding reveals a linear, non-branched ratchet mechanism for the nucleotide addition cycle in which translocation is one of the rate-limiting steps. We further determined all the major on- and off-pathway kinetic parameters in the elongation cycle. The resulting translocation energy landscape shows that the off-pathway states are favored thermodynamically but not kinetically over the on-pathway states, conferring the enzyme its propensity to pause and furnishing the physical basis for transcriptional regulation