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, where she was a fellow of the 
Helen Hay Whitney Foundation and a Lucille P. Markey Scholar. 
MY laboratory is particularly interested in the 
mechanisms that regulate the translation of 
messenger RNA (mRNA) into proteins. The infor- 
mation in mRNA is translated by a large RNA- 
protein complex, the ribosome. It has been 
known for some time that the ribosomes do not 
move along the mRNA at an even pace — that they 
pause at discrete places along the way. Why ribo- 
somes pause is not well understood but is 
thought to be due either to rare codons or to sec- 
ondary mRNA structures. 
In certain mRNAs the pausing of ribosomes is 
thought to cause them to slip and lose their place, 
such that a different protein is now translated. 
This slipping is known as ribosome frameshifting. 
We have been investigating why ribosomes pause 
during translation, and we would like to under- 
stand how cells can use ribosome pausing to regu- 
late the synthesis of particular proteins. 
To address these questions, we use a method 
that allows us to determine the distribution of 
ribosomes on an mRNA with single-nucleotide 
precision. In this way we can obtain a detailed 
picture of the translation process. Using this as- 
say, we have found that the ribosomes often 
pause directly over the initiation codon of the 
mRNA. This pausing by fully assembled ribo- 
somes appears to represent a slow step in eukar- 
yotic protein initiation that had not been previ- 
ously detected. 
By performing translation reactions in the pres- 
ence of inhibitors that block distinct steps in poly- 
peptide initiation and then examining the posi- 
tion of the resulting paused ribosomes, we have 
narrowed down this major slow step to one of two 
points in the initiation pathway. We are continu- 
ing to characterize this intermediate in transla- 
tion initiation and are also investigating the possi- 
bility that this slow step in protein synthesis may 
be a point at which cells regulate translation of 
particular mRNAs. 
We are also interested in understanding how 
the availability of particular tRNA molecules (in- 
termediates in protein synthesis) affects ribo- 
some pausing during elongation of the nascent 
protein. To study this question, we have prepared 
translation extracts in which the tRNA molecules 
have been removed. By adding back different 
tRNA populations, we can now manipulate the 
levels of individual tRNAs. In this way we hope to 
determine how different tRNA species contribute 
to ribosome pausing and frameshifting. 
We are also studying how the attachment of 
ribosomes to the endoplasmic reticulum mem- 
brane affects their movement along mRNAs en- 
coding secreted proteins. The synthesis of these 
proteins designed for export outside the cell be- 
gins when ribosomes, free in the cytoplasm, initi- 
ate translation on the mRNAs. A common feature 
of these secretory proteins is the presence of a 
signal peptide, usually an amino-terminal exten- 
sion of 15-30 amino acids. A signal-recognition 
particle (SRP), a small cytoplasmic ribonucleo- 
protein, binds to the signal sequence emerging 
from the ribosome and arrests elongation tran- 
siently. This translational arrest is released after 
the ribosome-SRP complex interacts with a spe- 
cific component of the endoplasmic reticulum 
membrane, the SRP receptor. 
Concomitant with the resumption of protein 
synthesis, translocation of the nascent polypep- 
tide begins. All subsequent translation is carried 
out by ribosomes that are attached to the endo- 
plasmic reticulum membrane. 
We have found that the point at which mem- 
brane insertion of the nascent polypeptide begins 
is distinct for different proteins, which may re- 
flect differences in the size and structures of indi- 
vidual signal peptides. It also appears that certain 
features of mRNA structure that cause ribosomes 
to pause during translation in solution do not al- 
ways result in their pausing when attached to 
microsomal membranes. Thus it appears likely 
that mRNA secondary structure can be altered 
by attachment through ribosomes to these 
membranes. 
We are currently determining whether ribo- 
somes, following termination, are able to reini- 
tiate translation on microsomal membranes. If so, 
it would provide a molecular explanation for the 
"circular polysomes" that have long been ob- 
served attached to the endoplasmic reticulum. 
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