Now Johns Hopkins scientists have found that a structure inside a cell’s protein-making machinery performs an unexpected negative “enforcer” function in addition to its known “positive” roles as protector and promoter of protein production. The research, which focuses on the odd chemical “cap” of messenger RNA, is described in the September 24 issue of Molecular Cell.
The team revealed that the cap — a baroque structure made of modified RNA constituents joined head to head rather than the usual head-to-tail arrangement found in the rest of the messenger RNA — prevents the manufacture of faulty proteins early on, at the very start of the complex process of protein manufacture, when messenger RNA delivers the genetic code from DNA to the decoding machine, which later translates it into proteins.
The team began its study in yeast by separating out all the various parts of the protein-making machinery. The scientists then modified some messenger RNA by lopping off its chemical “cap,” and left the rest intact so that when they reconstituted the machine in test tubes, they could compare the behaviors of capped and uncapped versions of messenger RNA.
Because they added a radioactive atom to the messenger RNA and fluorescent tags to the other proteins required for protein manufacture, the team could evaluate mRNA recruitment under various conditions — with and without a cap, for instance, and in the presence and absence of various other factors. Because the cap was known to play two positive roles in the protein manufacturing process, the researchers were surprised to see that the cap-less mRNA bound even better than the capped mRNA to protein builders called ribosomes.
“This was strange, because the cap of messenger RNA is supposed to be both a stabilizer and a stimulator of binding,” says Jon Lorsch, Ph.D., professor of biophysics and biophysical chemistry, Johns Hopkins University School of Medicine.
Further studies revealed that when the cap was removed, the mRNA bound efficiently but indiscriminately, even when all the necessary other proteins were not present. In this case, normal proteins could not be made. The cap appears to prevent the mRNA from interacting with the ribosome unless all of the required proteins are present and accounted for, according to Lorsch. When all of the factors are present, the cap stimulates binding. When they aren’t there, the cap inhibits binding.
“The cap is an enforcer,” Lorsch says, “and the only pathway it’s going to allow the mRNA down is the right one. It’s preventing it from going down this wrong pathway that leads to aberrant products.”
The work offers evidence, Lorsch says, that “biology is maybe more about preventing the negatives from happening than about promoting the positives.”
“The key,” he adds, “is keeping all the many possible incorrect complexes from forming so that the right one can be made. If you think about diseases such as sickle cell anemia, Alzheimer’s and cancer, they are examples of the wrong things taking place when they shouldn’t; they indicate a breakdown in prevention mechanisms.”
The study was supported by the National Institute of General Medical Sciences at the National Institutes of Health.
Johns Hopkins authors of the study, in addition to Lorsch, are Sarah F. Mitchell, Sarah E. Walker and Mikkel A. Algire. Other authors are Eun-Hee Park and Alan G. Hinnebusch of the National Institute of Child Health and Human Development.