Influence ofGuanine Nucleotides andElongation Factors onInteraction ofRelease Factors withtheRibosome

Releaseofformylmethionine fromthe reticulocyte ribosomalsubstrate, f(3HjMet-tRNA ribo- some,ispromotedbyreticulocyte release factor (RF).The initial rateofthisreaction isstimulated byGTP butin- hibited byGDPCP.FormationofanRF.UA(3HjA2-ribo- somecomplexisameasureofthebinding ofreticulocyte RF totheribosome, andtherecovery ofthiscomplexis increased byGDPCP and,toalesser extent, GTP.These studies suggest thatGTP isinvolved intheinitial associa- tionofRF withtheribosomeandthathydrolysis ofthe y-phosphate oftheguaninenucleotide isrequired ata subsequent rate-limiting step.Theribosomal-dependent fMet-tRNA hydrolysis andGTPaseactivities ofreticulocyte RF areinhibited whenelongation factor (EF)-2isbound totherespective ribosomal substrate inthepresence of fusidic acidandGDP.When EF-Gisboundtothef(3H)- Met-tRNAAUG ribosome substrate withfusidic acidand GDP,thefMet-tRNA hydrolysis activity ofEscherichia coli RF-1andRF-2isalsoinhibited. Thebinding ofreticulo- cyteRF andE.coliRF-1orRF-2totheirrespective ribo- somesisprevented whenfusidic acid.EF-2/EF-G'GDP ribosome complexes areused. Peptide chain termination hasbeenstudied inmammalian extracts byslight modifications oftheformylmethionine (fMet) release assaydescribed forbacterial extracts (1,2). Release off(3H)Met fromreticulocyte f('H)Met-tRNA- ribosome substrates isdirected byrandomly ordered poly- nucleotides orbytetranucleotides ofdefined sequence con- taining theterminator codons (UAA,UAG,orUGA).This hydrolysis requires reticulocyte release factor, RF,andis stimulated byGTP.AnRFfraction fromrabbit reticulocytes waspurified several hundred-fold andfoundtopossess a ribosomal dependent GTPasethatisstimulated byUAAA (3). Furthermore, this GTPaseisstimulated byfusidic acid, a knownantibiotic inhibitor oftheribosomal-dependent GTPaseactivities ofprokaryotic andeukaryotic elongation factors (EF-GandEF-2)(3-6). Fusidic acidinhibits this intermediate step ofprotein biosynthesis indirectly bystabiliz- ingtheEF-G/EF-2- GDP ribosome complexes (7-9). These stable complexes havebeenuseful instudying theinteraction ofbacterial EF-Gandaminoacyl-tRNA withtheribosome. Recentstudies haveshownthatbinding ofEF-Gtothe bacterial ribosome prevents bothnonenzymatic ribosomal binding ofaminoacyl-tRNA andthatcatalyzed byEF-Tu (10-13); conversely, binding ofaminoacyl-tRNA totheribo- somewithorwithout EF-Tuprevents subsequent association ofEF-Gwiththese ribosomes (12, 14). Similarly, inmam- malian extracts, EF-2boundtotheribosome prevents the ribosomal binding ofaminoacyl-tRNA catalyzed byEF-1(15). Since theribosomal-dependent GTPaseassociated with reticulocyte RF isaffected byfusidic acid, theribosomal interactions oftherelease factors andelongation factors havebeenexamined inthis report. Formation ofareticulocyte RF UA(8H)A2. ribosome complex hasbeenused, inaddition toreported assays forpeptide chain termination (1, 2,3,18), toinvestigate theeffects ofguanine nucleotides andelongation factors ontheinteraction ofRFwith theribosome.