Positive and negative effects of the major mammalian messenger ribonucleoprotein p50 on binding of 40 S ribosomal subunits to the initiation codon of beta-globin mRNA.
p50, the major core protein bound to mammalian mRNAs, has been reported to stimulate translation at low p50/mRNA ratios and inhibit translation at high p50/mRNA ratios. This study aims to address the molecular mechanisms underlying these phenomena using the in vitro assembly of 48 S preinitiation complexes from fully purified translational components in the presence or absence of p50 as analyzed by the toeprint assay. With limited concentrations of eIF2, eIF3, and eIF4F, p50 (but not pyrimidine tract-binding protein, which was taken for comparison) strongly stimulates formation of the 48 S preinitiation complexes with beta-globin mRNA. This stimulation is observed when just a few molecules of p50 are bound per molecule of the mRNA. When the amount of p50 in solution is increased over some threshold p50/mRNA ratio, a remarkable repression is observed that can still be relieved by adding more eIF2 and eIF4F. At even higher concentrations of p50, the inhibitory effect becomes irreversible. The threshold ratio depends upon the extent of secondary structure of the 5'-untranslated region linked to the beta-globin coding region. Chemical probing has confirmed that the binding of p50 to mRNA involves only the sugar-phosphate backbone of the mRNA leaving nucleotide bases free for interaction with other messenger ribonucleoprotein (mRNP) components. These data are best compatible with the functional role of p50 as a "manager" of mRNA-protein interactions in mammalian mRNPs.
Pisarev
Andrey V
AV
A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, 119899 Moscow, Russia.
Skabkin
Maksim A
MA
Thomas
Adri A
AA
Merrick
William C
WC
Ovchinnikov
Lev P
LP
Shatsky
Ivan N
IN
D000818:Animals; D001483:Base Sequence; D018387:Codon, Initiator / Q000235:genetics; D017931:DNA Primers; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism; D039621:Eukaryotic Initiation Factor-3; D039562:Eukaryotic Initiation Factor-4F; D005914:Globins / Q000235:genetics*; D008322:Mammals; D010448:Peptide Initiation Factors / Q000378:metabolism; D011132:Polyribosomes / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics*; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism*
D018387:Codon, Initiator; D017931:DNA Primers; D015852:Eukaryotic Initiation Factor-2; D039621:Eukaryotic Initiation Factor-3; D039562:Eukaryotic Initiation Factor-4F; D010448:Peptide Initiation Factors; D012333:RNA, Messenger; D005914:Globins