Researchers have discovered what is believed to be a novel method in yeast for governing gene expression at the end, rather than the beginning, of transcription, the process of reading DNA to make RNA. They report in the September 20 issue of the journal Nature that a protein called Nrd1 helps an enzyme, RNA polymerase II, recognize an as yet unknown "stop sign" for certain genes.
Because Nrd1 also controls its own transcription, the finding may help researchers understand a similar process HIV uses to hijack a cell's replicating machinery, say the scientists. While a similar mechanism is known in bacteria, this is the first example in yeast, the simplest organism whose cells have nuclei, says Jeffry [sic] Corden of the Johns Hopkins School of Medicine.
"Nrd1 doesn't just flag a new stop sign, it actively takes part in controlling gene expression -- the process of interpreting genetic information to make proteins," says Corden, a professor in the department of molecular biology and genetics, part of the school's Institute for Basic Biomedical Sciences. "Having an example of this mechanism in yeast is valuable because it will let us work out some of the fundamental mechanisms at play in regulating gene expression at the level of termination."
RNA polymerase II, which transcribes all protein-encoding genes, is very similar in organisms ranging from yeast to humans. While many details of transcription are unknown, it's clear that proper termination is crucial for the correct manufacture of proteins from genes, says Corden.
"These termination signals are sort of like traffic signs -- if you remove a stop sign, you're going to have an accident," he says. "When this enzyme picks a gene to transcribe, you don't want it transcribing into the next gene."
Corden and collaborators from Johns Hopkins and the University of Wisconsin Medical School already knew that Nrd1 binds to a region of RNA polymerase II called the C-terminal domain (CTD), which sticks out of the globular enzyme like a handle.
Now, in yeast that produce disabled Nrd1, the scientists have discovered that polymerase runs some stop signs, reading through the ends of certain genes until it reaches a different kind of stop signal. The research at Hopkins was funded by the National Science Foundation.
RNA polymerase II transcribes the 30,000 or so genes present in human cells by binding to an initiation region on the DNA, then "reading" the DNA. A strand of RNA is created as the enzyme moves down the gene. Eventually, the polymerase reaches a stop signal at the end of the gene.
In a 15-year effort to understand how the enzyme works, Corden and his lab have been hunting for genes and proteins that interact with the CTD. They identified a protein called SCAF8 in humans that binds to the CTD, but, lacking a good way to study its function, moved to yeast, where they began examining its counterpart, Nrd1.
To learn about Nrd1's normal function, the Hopkins scientists created yeast mutants with a temperature-sensitive Nrd1 gene. In these mutants, above a certain temperature the gene produces an altered form of Nrd1 that can't bind the CTD and hence can't affect its target genes.
Because the scientists believed Nrd1 limited transcription of certain genes, they then used microarray analysis, commonly known as "gene chips," to hunt for gene products that appeared to increase without Nrd1 to control their transcription.
When the researchers were unable to identify a common function for the genes that seemed to increase, they used the yeast genome database, which contains information about all 6,000 yeast genes, to find a unifying thread. All of these seemingly increased genes were right next to genes for a certain class of RNA molecules, called snoRNAs. Instead of coding for proteins themselves, these RNA molecules guide enzymes to modify other RNAs.
"We realized we were not looking at these genes increasing in the absence of Nrd1, but instead we were seeing read-throughs from the adjacent genes, which additional studies proved," says Corden.
While snoRNA genes seem to rely on a Nrd1 stop sign, the Nrd1 gene itself seems to have two stop signs: one is triggered only when Nrd1 is present, and the other, farther away, is triggered when Nrd1 is not binding to the CTD.
"We noticed that when Nrd1 is functioning properly, level of the protein is pretty low," says Corden. "But when we have non-functional Nrd1, suddenly there's all this Nrd1 protein. So Nrd1 autoregulates - it gets its own level just right. It is the first evidence for this type of autoregulation of cellular genes at the level of termination in yeast."
Nrd1's human counterpart, SCAF8, is unlikely to recognize the same DNA sequence, which the scientists are still trying to identify, or to be involved with termination of the same genes, says Corden. Ongoing studies will re-examine SCAF8 and also clarify some of the steps involved in nrd1-regulated termination.
Other authors are Nicholas Conrad, formerly at Hopkins and now at Yale, and Eric Steinmetz and David Brow of Wisconsin.
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