Mapping the ribosomal RNA neighborhood of protein L11 by directed hydroxyl radical probing.
Ribosomal protein L11 is a highly conserved protein that has been implicated in binding of elongation factors to ribosomes and associated GTP hydrolysis. Here, we have analyzed the ribosomal RNA neighborhood of Escherichia coli L11 in 50 S subunits by directed hydroxyl radical probing from Fe(II) tethered to five engineered cysteine residues at positions 19, 84, 85, 92 and 116 via the linker 1-(p -bromoacetamidobenzyl)-EDTA. Correct assembly of the L11 derivatives was analyzed by incorporating the modified proteins into 50 S subunits isolated from an E. coli strain that lacks L11 and testing for previously characterized L11-dependent footprints in domain II of 23 S rRNA. Hydroxyl radicals were generated from Fe(II) tethered to L11 and sites of cleavage in the ribosomal RNA were detected by primer extension. Strong cleavages were detected within the previously described binding site of L11, in the 1100 region of 23 S rRNA. Moreover, Fe(II) tethered to position 19 in L11 targeted the backbone of the sarcin loop in domain VI while probing from position 92 cleaved the backbone around bases 900 and 2470 in domains II and V, respectively. Fe(II) tethered to positions 84, 85 and 92 also generated cleavages in 5 S rRNA around helix II. These data provide new information about the positions of specific features of 23 S rRNA and 5 S rRNA relative to protein L11 in the 50 S subunit and show that L11 is near highly conserved elements of the rRNA that have been implicated in binding of tRNA and elongation factors to the ribosome.
Holmberg
L
L
Center for the Molecular Biology of RNA, Sinsheimer Laboratories, University of California, Santa Cruz, CA, 95064, USA.
Noller
H F
HF
D019943:Amino Acid Substitution; D001483:Base Sequence; D001665:Binding Sites; D002614:Chelating Agents; D003545:Cysteine; D004492:Edetic Acid / Q000031:analogs & derivatives; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D017665:Hydroxyl Radical; D007501:Iron; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D012338:RNA, Ribosomal, 23S / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012269:Ribosomal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism / Q000648:ultrastructure
D002614:Chelating Agents; D012338:RNA, Ribosomal, 23S; D011994:Recombinant Proteins; D012269:Ribosomal Proteins; C027624:ribosomal protein L11; D017665:Hydroxyl Radical; C042007:1-(4-bromoacetamidobenzyl)EDTA; D004492:Edetic Acid; D007501:Iron; D003545:Cysteine