In vitro assembly of yeast 5S rRNA and a fusion protein containing ribosomal protein L5 and maltose binding protein.
Binding of yeast ribosomal protein L5 with 5S rRNA has long been considered a promising model for studying molecular mechanisms of protein-RNA interactions. However, in vitro assembly of a ribonucleoprotein (RNP) complex from purified yeast ribosomal protein L5 (also known as L1, L1a, or YL3) and 5S rRNA proved to be difficult, thus limiting the utility of this model. In the present report, we present data on the successful in vitro assembly of a RNP complex using a fusion (MBP-L5) protein consisting of the yeast ribosomal protein L5 fused to the carboxyl terminus of the E. coli maltose-binding protein (MBP). We demonstrated that: 1) the MBP-L5 protein binds yeast 5S rRNA but not 5.8S rRNA in vitro; 2) the MBP protein itself does not bind yeast 5S rRNA; 3) formation of the RNP complex is proportional to the concentration of MBP-L5 protein and 5S rRNA; and 4) the MBP moiety of the fusion protein in the RNP complex can be removed with factor Xa. The electrophoretic mobility of the resultant RNP complex is indistinguishable from that of L5-5S rRNA complex isolated from the ribosome. Using this new experimental approach, we further showed that the RNA binding capability of a mutant L5 protein is decreased by 60% compared to the wild-type protein. Additionally, the mutant RNP complex migrates slower than the wild-type RNP complex suggesting that the mutant RNP complex has a less compact conformation. The finding provides a probable explanation for an earlier observation that the 60S ribosomal subunit containing the mutant protein is unstable.
Pakhomova
O N
ON
Department of Biochemistry, The University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78284-7760 USA.
Yeh
L C
LC
Monette
J
J
Lee
J C
JC
D018528:ATP-Binding Cassette Transporters; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D009154:Mutation; D011485:Protein Binding; D012331:RNA, Fungal / Q000235:genetics / Q000378:metabolism*; D012341:RNA, Ribosomal, 5S / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012261:Ribonucleoproteins / Q000235:genetics / Q000378:metabolism; D012269:Ribosomal Proteins / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*
D018528:ATP-Binding Cassette Transporters; D002352:Carrier Proteins; D017931:DNA Primers; D029968:Escherichia coli Proteins; D005656:Fungal Proteins; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D012331:RNA, Fungal; D012341:RNA, Ribosomal, 5S; D011993:Recombinant Fusion Proteins; D012261:Ribonucleoproteins; D012269:Ribosomal Proteins; C461599:maltose transport system, E coli; C029742:ribosomal protein L5