A pre-translocational intermediate in protein synthesis observed in crystals of enzymatically active 50S subunits.
The large ribosomal subunit catalyzes peptide bond formation during protein synthesis. Its peptidyl transferase activity has often been studied using a 'fragment assay' that depends on high concentrations of methanol or ethanol. Here we describe a version of this assay that does not require alcohol and use it to show, both crystallographically and biochemically, that crystals of the large ribosomal subunits from Haloarcula marismortui are enzymatically active. Addition of these crystals to solutions containing substrates results in formation of products, which ceases when crystals are removed. When substrates are diffused into large subunit crystals, the subsequent structure shows that products have formed. The CC-puromycin-peptide product is found bound to the A-site and the deacylated CCA is bound to the P-site, with its 3prime prime or minute OH near N3 A2486 (Escherichia coli A2451). Thus, this structure represents a state that occurs after peptide bond formation but before the hybrid state of protein synthesis.
Schmeing
T Martin
TM
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520-8114, USA.
Seila
Amy C
AC
Hansen
Jeffrey L
JL
Freeborn
Betty
B
Soukup
Juliane K
JK
Scaringe
Stephen A
SA
Strobel
Scott A
SA
Moore
Peter B
PB
Steitz
Thomas A
TA
D000438:Alcohols / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D001665:Binding Sites; D002384:Catalysis; D003460:Crystallization; D004926:Escherichia coli; D019614:Haloarcula marismortui; D008958:Models, Molecular; D009239:N-Formylmethionine / Q000378:metabolism; D014176:Protein Biosynthesis; D011487:Protein Conformation; D021122:Protein Subunits; D011691:Puromycin / Q000378:metabolism; D012270:Ribosomes / Q000737:chemistry* / Q000378:metabolism*; D012996:Solutions; D012997:Solvents / Q000378:metabolism; D014961:X-Ray Diffraction
D000438:Alcohols; D001426:Bacterial Proteins; D021122:Protein Subunits; D012996:Solutions; D012997:Solvents; D009239:N-Formylmethionine; D011691:Puromycin