Like a DVD player, RNA polymerase (RNAP) comes with pause functionality, but exactly how the enzyme enters and leaves the paused state has remained unclear. Just looking at the machinery, the RNAP-DNA complex, is about as informative as peering around the DVD player’s slot-load tray. A better understanding of the pause function requires a little mechanistic dissection.

Noticing that RNAP isn’t labeled “no user-serviceable parts inside,” scientists based at the University of Wisconsin-Madison applied their kinetic analysis and modeling tools to RNAP’s elongation complex. These scientists, led by Robert Landick, PhD, Charles Yanofsky professor of biochemistry and bacteriology, found that the pause process involves several biological players, which together create a barrier to prevent escape from paused states. The process also causes a modest conformational shift that makes RNAP “stumble” in feeding DNA into its reaction center, temporarily stopping it from making RNA.

“We also found that transcriptional pausing makes RNAP loosen its grip and backtrack on the DNA while paused,” noted Landick. “Together, these results provide a framework to understand how the process is controlled by certain conditions and regulators within cells.”

Additional details appeared January 8 in the journal eLife, in an article titled, “The elemental mechanism of transcriptional pausing.” According to this article, the mechanistic basis of RNAP’s pause functionality could provide a framework to understand how pausing is modulated by sequence, cellular conditions, and regulators. That is, a mechanistic understanding of transcriptional pausing could improve our understanding of how RNAP could be taken offline by new drugs, including drugs for conditions such as Clostridium difficile infections and tuberculosis.

“We report,” the authors of the eLife article wrote, “a mechanistic dissection that establishes the elemental pause signal (i) is multipartite; (ii) causes a modest conformational shift that puts g-proteobacterial RNAP in an off-pathway state in which template base loading but not RNA translocation is inhibited; and (iii) allows RNAP to enter pretranslocated and one-base-pair backtracked states easily even though the half-translocated state observed in paused cryo-EM structures rate-limits pause escape.”

Gene expression occurs when the information contained in DNA is used to produce functional gene products such as proteins and other molecules. The process has two stages. In the first stage, called transcription, RNAP reads the information in a strand on DNA, which is then copied into a new molecule of messenger ribonucleic acid (mRNA). In the second stage, the molecule then moves on to be processed or translated.

However, to help control gene expression levels, transcriptional pausing by RNAP can occur between the two stages, providing a kind of “hold” whereby transcription may be terminated or modulated.

“A consensus pause sequence that acts on RNAPs in all organisms, from bacteria to mammals, halts the enzyme in an elemental paused state from which longer-lived pauses can arise,” explained Landick, the article’s senior author. “As the fundamental mechanism of this elemental pause is not well defined, we decided to explore this using a variety of biochemical and biophysical approaches.”

He added that the insights gained in the current study could aid future efforts to design synthetic genes, for example, to direct the pausing behavior of RNAP in a way that yields desired outputs from genes. It could also help our understanding of how certain drugs, known as RNAP inhibitors, target the enzyme.

“For now, we would like to try and generate structures of paused transcription complexes obtained at a series of time intervals,” Landick concluded. “This would allow us to see exactly how parts of the enzyme move as it enters and leaves the paused state.”

Another recent study looked at RNAP and transcriptional pausing from a different perspective. This study, conducted by researchers at the Centre for Genomic Regulation (CRG) in Barcelona, Spain, in collaboration with scientists at the University Pompeu Fabra (UPF) and the Helmholtz Center Munich, described a novel modification of the carboxyl terminal domain of RNAP, namely the deimination of an arginine, by the enzyme PADI2. This modification, the scientists reported, allows the polymerase to transcribe genes relevant for cancer cell growth.

This work appeared November 21 in the journal Molecular Cell, in an article titled, “Arginine Citrullination at the C-Terminal Domain Controls RNA Polymerase II Transcription.” According to the authors of this article, the newfound mechanism might point to drugs capable of targeting just the particular action of PADI2 on RNA polymerase needed for tumor progression without globally blocking the enzyme.

“We observed that breast cancer cells need a particular modification to express a set of genes required for cellular proliferation and tumor progression,” explained Priyanka Sharma, PhD, a CRG researcher and the first author of Molecular Cell paper. “This modification allows the enzyme RNA polymerase II to overcome a pausing barrier and to continue to transcribe these genes.”

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