University of Pittsburgh study sheds light on how the cell decides precisely where to start copying a gene

The DNA helix, found within all living cells, carries the template or code for the production of the many different proteins essential for life. But how does the cell know exactly where to start copying the DNA?

The first stage of protein production is called transcription, in which a portion of the DNA strand is selected to be unraveled and copied. To initiate transcription, molecular machinery assembles at a defined region of the genome and melts the double stranded DNA, leaving a region of unwound DNA known as a “transcription bubble.” The machinery must then travel down the unwound strand, scanning for a place to begin, and initiate the process of transcription.

To better understand how a transcription start site is selected, researchers led by Craig D. Kaplan at the University of Pittsburgh carried out detailed studies on a common species of yeast, Saccharomyces cerevisiae, used since ancient times in brewing, winemaking and baking. 

Their results, published October 2021 in the journal eLife Sciences, provide new insights into how far a certain protein scans in the promoter region, and how often it initiates transcription. The lead author is Tingting Zhao, currently at the Genomics and Bioinformatics Hub, Brigham and Women’s Hospital, Harvard Medical School in Boston. 

Transcription factor TFIIH

In eukaryotic organisms (those with a distinct cell nucleus), transcription is carried out by a protein known as RNA polymerase POL II. 

For the transcription to start, POL II requires help from at least five specialized proteins called general transcription factors. One of these transcription factors, known as TFIH, already was thought to play a crucial role in finding the start site for transcription. 

However, the exact mechanism of transcription starter site selection (TSS) was not well understood. Kaplan’s team has made progress in this area by studying a protein called Ssl2.

Earlier studies suggested that the transcription factor TFIIH drives the DNA scanning process in the yeast organism using ATP (adenosine triphosphate). The hypothesis has been that at the beginning of scanning, a pre-initiation complex (PIC) assembles upstream of the transcription start site then Ssl2 within TFIIH facilitates interrogation of base pairs for a start site.

Key role of Ssl2

Kaplan’s team used both optical and magnetic tweezer-based single molecule analysis to aid their investigation. Both studies agreed that the likely agent for the PIC activity is an ATP-dependent enzyme called Ssl2 that acts within the transcription factor TFIIH. Ssl2 is the homolog in yeast of the ATP-dependent DNA helicase enzyme known as XPB that drives transcription in humans. 

The Kaplan lab has identified residues on Ssl2, which shift where transcription begins. This could lead to an explanation of the mechanism by which transcription start sites are selected. 

“We propose that the outcome of promoter scanning is determined by two functional networks,” the paper concludes. “The first [is] Pol II activity and factors that modulate it to determine initiation efficiency within a scanning window, and the second being Ssl2/TFIIH and factors that modulate scanning processivity to determine the width of the scanning widow.” 

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Tingting Zhao et al. Ssl2/TFIIH function in transcription start site scanning by RNA polymerase II in Saccharomyces cerevisiae. eLife Sciences (2021).  DOI: https://doi.org/10.7554/eLife.71013