J. Biol. Chem., Vol. 263, Issue 15, 7164-7169, 05, 1988
Shifts in configuration of the 5'-noncoding region of a mouse messenger RNA under translational control
ST Chitpatima and G Brawerman
Department of Biochemistry, Tufts University Health Sciences Schools, Boston, Massachusetts 02111.
Several major mRNA species of mouse and other mammalian cells occur both as
small untranslated ribonucleoprotein particles and as functional molecules
associated with ribosomes in polysomes. One of these, that codes for a
21-kDa polypeptide, was analyzed with respect to distribution of sites
accessible to RNase T1 in the 5'-noncoding region. This region, which is
about 100 nucleotides long, contains several sites that are highly
sensitive to the enzyme, as well as many G residues not susceptible to
cleavage. The distribution of highly sensitive sites was compared in the
active and inactive states of the P21 mRNA present in cytoplasmic extracts
by subjecting the extract to limited nuclease digestion followed by
separation of partially fragmented polysomes from free messenger
ribonucleoprotein particles. The mRNA in polysomes contained two highly
sensitive sites, one near the 5' terminus and the other in the middle of
the region, next to a sequence potentially capable of Shine-Dalgarno
interaction. The untranslated molecules lacked the 5'-proximal site but had
several highly accessible sites not present in the active molecules. The
initiation AUG showed little accessibility both in polysomes and in
messenger ribonucleoproteins. Both forms were quite different from the
deproteinized mRNA with respect to distribution of nuclease-sensitive
sites. Our results indicate that interaction of the mRNA with cytoplasmic
factors strongly affects its conformation in the 5'- noncoding region and
that a particular conformation may be important for effective interaction
with ribosomal particles during polypeptide chain initiation.
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