Genetics:
Protein Reverses Chromatin Engineering

Biological Chemistry:
Molecule Implicated in Transcription Termination

Structural Biology:
DNA Splicing Enzyme Observed in Action

Scientific Symposium:
Fashions Change in Modeling Disease

Chronic Periodontitis Differs at the Microbial Level in Populations Worldwide

Brain Structure for Reward and Punishment Smaller in Cocaine Addicts

Brigham Celebrates 50th Anniversary of First Human Organ Transplant

Beth Israel Assumes Academic Oversight of Mass. Mental Health Faculty

Joslin Names Conley Chairman of the Board

Academic Officer Tapped for HMI Dubai Project

Macklis Receives Javits Neuroscience Investigator Award

Global Citizen Award Goes to Bill Moyers

HMS Family Health Guide Published in Paperback

New Appointments to Full Professor

Honors and Advances

Community Celebrates a Child's First Laugh

Front Page

Molecule Implicated in Transcription Termination

DNA makes RNA makes protein. Sounds simple enough, yet decades since this dogma was posited, fundamental questions remain. Perhaps the most intractable is not how the process starts, but what makes the first step--transcription--stop. While specific codons indicate where protein synthesis should end, there are no flags built into DNA to signal when messenger RNA should be terminated. So what does prevent RNA polymerase from barreling down the helix like a runaway train, transcribing gene after gene?

Termination of transcription is a Rat1 race. After polyA tails are added to growing mRNA chains, polyA factors vacate the C-terminal domain (CTD) of the RNA polymerase (RNA pol II), making room for recruitment of Rtt103, Rai1, and Rat1. The nuclease Rat1 then begins to digest the growing mRNA. Though the polymerase continues synthesis, Rat1 gives chase, and once it catches up to the polymerase catalytic domain, the entire complex detaches from DNA, terminating transcription. These findings are reported by (below, from left) Lidia Vasilieva, Minkyu Kim, and Stephen Buratowski. (Image by Jeff Cleary; Photo by Graham Ramsay)

At last, there may be an explanation. In the Nov. 25 Nature (see also companion story), Stephen Buratowski, HMS professor of biological chemistry and molecular pharmacology, and colleagues reveal several new components that are shunted onto the polymerase. One of them, a ribonuclease called Rat1, appears to join the complex as a caboose, derail it from the DNA, and terminate transcription.

Telling Tails

To isolate potential termination factors, research fellow Minkyu Kim capitalized on the properties of repeated motifs found in the C-terminal domain of yeast RNA polymerase II (RNApII). When phosphorylated, these repeats bind to a variety of proteins, including those involved in polyadenylation and termination. By passing yeast extract over a matrix of the repeats, Kim isolated a novel RNApII binding partner, shown by mass spectrometry to be Rtt103. Could this be the terminator?

"We also found the ribonuclease Rat1 and its cofactor Rai1. This got us pretty excited because we knew the ribonuclease might give us a way to connect the dots between polyadenylation and termination."

Not quite. Though termination of transcription is essential for survival, Kim found that yeast mutants lacking the protein were still viable. Other clues suggested that Buratowski and colleagues were on the right track, however. A collaboration with Jack Greenblatt's lab at the University of Toronto revealed that yeast lacking Ctk1, a component of the kinase that phosphorylates those C-terminal repeats, need Rtt103 to survive. "Initially, we weren't very thrilled about pulling out Rtt103," said Buratowski, "but we thought it may lead us to other, more interesting proteins."

Again using affinity purification, this time with Rtt103 as bait, Kim isolated several more proteins. "We found some known RNA polymerase components, which wasn't very surprising, but then we also found the ribonuclease Rat1 and its cofactor Rai1. This got us pretty excited because we knew the ribonuclease might give us a way to connect the dots between polyadenylation and termination," said Buratowski.

Tying Up Loose Ends

To test this idea, postdoctoral fellow Lidia Vasilieva introduced into yeast a Rat1 that is only partially active. She then examined how RNA was processed. Vasilieva found that neither RNA cleavage nor polyadenylation were affected. But when she used the polymerase chain reaction to quantify the RNA being produced, she found that sequences downstream of the polyA signal were abundant in yeast with limited Rat1 activity. This suggested that Rat1 is needed to degrade the RNA made after the polyadenylation signal.

But does Rat1 play a role in termination? If it does, then it should preferentially bind to RNApII at the 3' end of genes. To test this, Kim, in collaboration with Oliver Rando, a fellow at Harvard's Bauer Center for Genomics Research, used a chromatin immunoprecipitation (ChIP) assay in which RNApII and associated proteins are cross-linked to DNA and immunoprecipitated. Then the bound DNA is identified, revealing which DNA sequences are bound by which protein.

The ChIP experiments showed that Rat1, Rai1, and Rtt103 all preferentially bound to DNA near the 3' end of genes, where termination occurs. But the telling experiment came when mutant yeast expressing partially active Rat1 were used. Normally, RNApII is evenly distributed along an actively transcribed gene, except at the 3' end, where the polymerase terminates and falls off the DNA. ChIP experiments on wild-type yeast confirmed this kind of distribution. But results from the Rat1 mutants showed that the polymerase density at the 3' end was similar to that upstream, indicating that termination was failing.

Altogether, the experiments suggest the following sequence of events (see figure). RNA polymerase, complete with polyadenylation factors at its C-terminal domain, travels along a gene synthesizing mRNA. Once the polyA signal gets incorporated, the chain gets cleaved, the polyadenine added, and the polyadenylation factors vacate. This allows Rat1 to bind, and now, being in close proximity to the new head of the RNA, the nuclease begins to digest it. A race then ensues. RNApII still works its way along the DNA, but it cannot synthesize RNA as fast as Rat1 digests it, and when the nuclease catches up to the catalytic domain of the polymerase, it somehow kicks the complex off the DNA, terminating transcription. "We are not exactly sure how the mechanics of this last step operates, but we are working on it," said Buratowski.

--Tom Fagan