Interaction of yeast elongation factor 3 with polynucleotides, ribosomal RNA and ribosomal subunits.
In addition to the two usual eukaryotic elongation factors (EF-1 alpha and EF-2) fungal ribosomes need a third protein, elongation factor 3, for translation. EF-3 is essential for in vivo and in vitro protein synthesis. Functionally, EF-3 stimulates EF-1 alpha dependent binding of aminoacyl-tRNA to the ribosomal A site when E site is occupied by deacylated tRNA. EF-3 has intrinsic ATPase activity which is regulated by the functional state of the ribosome. EF-3 ATPase is activated by both 40S and 60S ribosomal subunits. However intact 80S ribosomes are needed for efficient activation of EF-3 ATPase. EF-3 appears to be an RNA binding protein with high affinity for polynucleotides containing guanosine rich sequences. To determine whether guanosine rich sequence of ribosomal RNA is involved in EF-3 binding, an antisense oligonucleotide dC6 was used to block EF-3 interaction with the ribosome. The oligonucleotide suppresses activation of EF-3 ATPase by 40S ribosomal subunit and not by the 60S or the 80S particles. Poly(U)-directed polyphenylalanine synthesis by yeast ribosomes is inhibited by dC6. To define the binding site of the oligonucleotide and presumably of EF-3 on 18S ribosomal RNA, hydrolysis of rRNA by RNase H was followed in the presence of dC6. These experiments reveal an RNase H cleavage site at 1094GGGGGG1099 sequence of 18S ribosomal RNA. This guanosine rich sequence of rRNA is suggested to be involved in EF-3 binding to yeast ribosome. Data presented in this communication suggest that the activity of EF-3 involved a direct interaction with the guanosine rich sequence of rRNA.
Kovalchuke
O
O
Department of Biochemistry, Medical College of Wisconsin, Milwaukee 53226, USA.
Chakraburtty
K
K
D005656:Fungal Proteins / Q000378:metabolism*; D010445:Peptide Elongation Factors / Q000378:metabolism*; D011119:Polynucleotides / Q000378:metabolism*; D012331:RNA, Fungal / Q000378:metabolism*; D012335:RNA, Ribosomal / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism* / Q000648:ultrastructure; D029701:Saccharomyces cerevisiae Proteins
D005656:Fungal Proteins; D010445:Peptide Elongation Factors; D011119:Polynucleotides; D012331:RNA, Fungal; D012335:RNA, Ribosomal; D029701:Saccharomyces cerevisiae Proteins; C111584:YEF3 protein, S cerevisiae