Translational Regulation 
Sandra L. Wolin, M.D., Ph.D. — Assistant Investigator 
Dr. Wolin is also Assistant Professor of Cell Biology at Yale University School of Medicine. She received her 
undergraduate degree in biochemistry from Princeton University and her M.D. and Ph.D. degrees from 
Yale University, where she worked with Joan Steitz. Her postdoctoral research was done with Marc 
Kirschner and Peter Walter at the University of California, San Francisco. 
CELLS use diverse molecular mechanisms for 
regulating the synthesis of proteins in re- 
sponse to environmental changes. Any of the 
many steps in the pathway by which the informa- 
tion stored in genes becomes converted into pro- 
teins can be subject to regulation. My laboratory 
is particularly interested in understanding the 
process by which messenger RNA (mRNA) be- 
comes translated into proteins. We wish to un- 
derstand fundamental aspects of this process, as 
well as how translation of certain proteins can be 
regulated in response to conditions outside the 
cell. 
The information in mRNA is translated into 
protein by a large RNA-protein complex, the ri- 
bosome. It has been known for some time that the 
movement of ribosomes along the mRNA is not 
linear with time. Rather, they pause at discrete 
places along the way. Why they do so is not well 
understood, but is thought to be due either to 
rare codons or to secondary structures in the 
mRNA. 
As a postdoctoral fellow in Peter Walter's labo- 
ratory, I devised a new method to determine the 
distribution of ribosomes on an mRNA with 
single-nucleotide precision. Using this assay, we 
have been able to detect and map discrete sites of 
ribosome pausing during translation in extracts. 
Two of these rate-limiting steps correspond to ini- 
tiation and termination of translation. Others oc- 
cur during elongation of the nascent protein. We 
are using this method to identify features of 
mRNA sequence or structure that result in ribo- 
some pausing. 
Surprisingly, we have found that the pausing 
causes a sort of traffic jam, in that ribosomes pile 
up behind each paused one. Under certain condi- 
tions, we have detected up to nine ribosomes 
piled up behind the leading one. As a result, the 
ribosomes become tightly packed, such that their 
centers are only 27 nucleotides apart. This tight 
packing along the mRNA forces the ribosomes in 
each cluster to orient themselves in a zigzag 
pattern. 
Although we have only detected ribosome 
stacking in cell extracts, we are interested in de- 
termining if this phenomenon occurs in intact 
cells. "Zigzag" or "helical" polyribosomes have 
been previously seen in the electron micrographs 
of certain cells, but their functional significance 
has been unclear. It seems likely that they repre- 
sent densely stacked ribosome clusters, caused by 
the pausing of the leading ribosome. 
We are also investigating the mechanism by 
which the translation of a secretory protein, insu- 
lin, is regulated in response to conditions outside 
the cell. Insulin is the major protein involved in 
regulating glucose metabolism in mammals. It is 
synthesized by the (8 cells of the pancreas, which 
are organized into clusters known as islets. When 
primary cultures of rat pancreatic islets are incu- 
bated in high concentrations of glucose, the syn- 
thesis of the insulin precursor molecule, proin- 
sulin, increases between 5- and 10-fold within 
minutes. During the first hour of glucose adminis- 
tration, this increased synthesis of insulin is 
largely due to more efficient translation of preex- 
isting mRNA. Although the phenomenon has 
been described for many years, little is known of 
the molecular mechanisms involved. 
We are using the above assay, in conjunction 
with biochemical fractionation, to probe the 
mechanism by which the translation of proinsu- 
lin mRNA is regulated in pancreatic islets. Our 
initial results indicate that preproinsulin transla- 
tion is regulated at both the initiation and elonga- 
tion levels. We hope that a better understanding 
of this phenomenon will be helpful in explaining 
how, in certain patients with diabetes, normal in- 
sulin production is impaired. 
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