Effects of two photoreactive spermine analogues on peptide bond formation and their application for labeling proteins in Escherichia coli functional ribosomal complexes.
The effect of two photoreactive analogues of spermine, N(1)-azidobenzamidino- (ABA-) spermine and N(1)-azidonitrobenzoyl- (ANB-) spermine, on ribosomal functions was studied in a cell-free system derived from Escherichia coli. In the dark, both analogues stimulated the binding of AcPhe-tRNA to poly(U)-programmed ribosomes, enhanced the stability of the ternary complex AcPhe-tRNA.poly(U).ribosome (complex C), and caused stimulatory and inhibitory effects on peptidyltransferase activity. ABA-spermine exhibited more pronounced effects than ANB-spermine. Each photoprobe was covalently attached after irradiation to both ribosomal subunits and also to free rRNA isolated from 70S ribosomes. Photolabeled complex C showed a reactivity toward puromycin, similar to that exhibited by complex C reacting reversibly with photoprobes free in solution. The distribution of the incorporated radioactivity among the ribosomal components was determined under two experimental conditions, one stimulating and the other inhibiting peptidyltransferase activity. Under both conditions, ABA-spermine was the strongest cross-linker. Upon stimulatory conditions, 14% of ABA-[(14)C]spermine cross-linked to complex C was bound to the protein fraction. The proteins primarily labeled were identified as S3, S4, L2, L3, L6, L15, L17, and L18. Upon inhibitory conditions, a higher percent of the incorporated radioactivity was found in ribosomal proteins, while the pattern of protein labeling was characterized by a remarkable decrease of cross-linked proteins L2, L3, L6, L15, L17. and L18 and by an increase of cross-linked proteins S9, S18, L1, L16, L22, L23, and L27. On the basis of these results and literature data, the involvement of spermine in the conformation and important functions of ribosomes is discussed.
Amarantos
I
I
Laboratory of Biochemistry, School of Medicine, University of Patras, GR-26500 Patras, Greece.
Xaplanteri
M A
MA
Choli-Papadopoulou
T
T
Kalpaxis
D L
DL
D001386:Azides / Q000378:metabolism / Q000494:pharmacology*; D001665:Binding Sites / Q000187:drug effects; D003432:Cross-Linking Reagents / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D004926:Escherichia coli / Q000201:enzymology / Q000378:metabolism* / Q000502:physiology; D010452:Peptide Biosynthesis / Q000187:drug effects* / Q000502:physiology; D010458:Peptidyl Transferases / Q000378:metabolism; D019920:Photoaffinity Labels / Q000378:metabolism / Q000494:pharmacology*; D014176:Protein Biosynthesis / Q000187:drug effects; D012360:RNA, Transfer, Phe / Q000378:metabolism; D012269:Ribosomal Proteins / Q000096:biosynthesis / Q000378:metabolism; D012270:Ribosomes / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism / Q000502:physiology*; D013096:Spermine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology*
D001386:Azides; D003432:Cross-Linking Reagents; D019920:Photoaffinity Labels; D012360:RNA, Transfer, Phe; D012269:Ribosomal Proteins; C068638:N(1)-(azidobenzamidino)spermine; C069888:(5-azido-2-nitrobenzoyl)spermine; D013096:Spermine; D010458:Peptidyl Transferases