The canonical view of termination of transcription holds that after releasing the mRNA, RNAP dissociates from the DNA. But researchers from Clemson University have demonstrated for the first time force plays a role in an alternative scenario.
The genome inside each of our cells is modelled by tension and torsion — due in part to the activity of proteins that compact, loop, wrap and untwist DNA — but scientists know little about how those forces affect the transcription of genes.
“There are a lot of mechanical forces at play all the time that we never consider, we have very little knowledge of, and they’re not talked about in textbooks,” said Laura Finzi, the Dr. Waenard L. Miller, Jr. ’69 and Sheila M. Miller Endowed Chair in Medical Biophysics at Clemson University.
Transcription is the process by which a cell makes an RNA copy of a segment of DNA. One type of RNA, called messenger RNA (mRNA), encodes information to make proteins required for the structure and functions of cells or tissues.
Role of Force
RNA polymerase (RNAP) is a type of protein that produces mRNA. It tracks processively along double helical DNA, untwists it to read the base pair sequence of only one strand and synthesizes a matching mRNA. Such “transcription” of a gene begins when RNAP binds to a “promoter” DNA sequence and ends at a “terminator” sequence where the mRNA copy is released. The canonical view of termination holds that after releasing the mRNA, RNAP dissociates from the DNA.
A team of researchers led by Finzi and including David Dunlap, research professor in the Clemson Department of Physics and Astronomy, have, for the first time, demonstrated how force plays a role in an alternative to canonical termination.
Using magnetic tweezers to pull RNAP polymerase along a DNA template, the researchers were able to show that upon reaching a terminator, bacterial RNA polymerase may remain on the DNA template and be pulled to slide backward to the same or forward to an adjacent promoter to start a subsequent cycle of transcription. Thus, the direction of force determines whether a segment of DNA may be transcribed multiple times or only once. Finzi and Dunlap report that this force-directed recycling mechanism can change the relative abundance of adjacent genes.
Force-directed sliding leads to repetitive transcription
a A diagram of the experimental setup for transcription against opposing force. b A representative recording of multiple rounds of transcription under opposing force includes a temporary roadblock-associated pause during transcription (shaded region 1), pauses at the terminator (shaded regions 2), RNAP temporarily roadblocked during backward sliding (shaded region 3), and pauses at the promoter prior to re-initiation (shaded regions 4). The inset shows data points corresponding to RNAP sliding back from the terminator in cycle 1. c On templates with a dual terminator sequence, the percentage of RNAP that slid backward was twice that on templates with single terminators. The total number of events are listed above each bar. d Opposing force (negative values) significantly raised the probability that the post-terminator complex slid toward the promoter from which the previous cycle of transcription initiated. The total number of events are listed above each bar. e RNAP sliding rates increased rapidly as opposing (–) or assisting (+) force increased from 0 to 0.7 pN but plateaued thereafter. The red center line denotes the 50th percentile, while the blue box contains the 25th to 75th percentiles of the dataset. The black whiskers mark the 5th and 95th percentiles, and values beyond these upper and lower bounds are considered outliers, marked with red crosses. The numbers of sliding events (N) are 128, 1086, 131, 104, 10, 4, 16, 5 for the -5, -2, -0.7, -0.2, 0.2, 0.7, 2, 5 pN force conditions, respectively. Source data are provided as a Source Data file.
Flipping Around
Furthermore, they found that the ability of a sliding RNAP requires the C-terminal domain of the alpha subunit to recognize a promoter oriented opposite to the direction of sliding. These subunits “allow it to stay on track, flip around and grab the other strand of the DNA double helix where another promoter might be,” she said. Indeed, with the alpha subunits deleted, flipping around to oppositely oriented promoters did not occur.
A thorough understanding of the molecular mechanisms that regulate transcriptional activity in the genome may identify therapeutic alternatives in which RNAP might be modified to suppress certain proteins and prevent disease.
Finzi said there might be locations in the genome where recycling is more frequent than others, but that is still unknown.
“My hope is that one day we will have a spatio-temporal map of forces acting on the genome at various times during the life cycle of various types of cells in our organism. Our research highlighting the effect of forces on the probability of repetitive transcription may then help predicting and plotting, in a heat map sort of way, the different levels of transcription of different genes,” Finzi said.
Source – Clemson University