Crosslinking of 4.5S RNA to the Escherichia coli ribosome in the presence or absence of the protein Ffh.
Radioactively labeled 4.5S RNA containing statistically distributed 4-thiouridine residues in place of normal uridine was prepared by T7 transcription. The ability of this modified 4.5S RNA to form a complex with the protein Ffh was demonstrated by a gel shift assay. The modified 4.5S RNA, with or without Ffh, was added to Escherichia coli ribosomes under various conditions, and crosslinking from the thiouridine residues was induced by irradiation at 350 nm. The crosslinked ribosomal components were analyzed by our standard procedures. Two clearly defined types of crosslinking were observed. The first was a crosslink to 23S rRNA, which was entirely dependent both on the presence of Ffh and a nascent protein chain in the 50S subunit. This crosslink was localized to nt approximately 2828-2837 of the 23S rRNA, from position 84 of the 4.5S molecule. The second type of crosslinking, to the 30S ribosomal subunit, was independent of the presence of Ffh, and was found both with vacant 70S ribosomes or isolated 30S subunits. Here the crosslink was localized to the 3'-terminal region of the 16S rRNA, from positions 29-50 of the 4.5S RNA. Cross-linking to ribosomal protein S1 was also observed. The known crystal structure of the protein Ffh/4.5S RNA fragment complex was extrapolated by computer modeling so as to include the whole 4.5S molecule, and this was docked onto the ribosome using the crosslinking data. The results are discussed in terms of multiple functions and binding sites of the 4.5S RNA.
Rinke-Appel
Jutta
J
Max-Planck-Institut für Molekulare Genetik, Berlin, Germany.
Osswald
Monika
M
von Knoblauch
Klaus
K
Mueller
Florian
F
Brimacombe
Richard
R
Sergiev
Petr
P
Avdeeva
Olga
O
Bogdanov
Alexey
A
Dontsova
Olga
O
D001483:Base Sequence; D001665:Binding Sites; D003432:Cross-Linking Reagents; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D046911:Macromolecular Substances; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011487:Protein Conformation; D012329:RNA, Bacterial / Q000737:chemistry* / Q000378:metabolism*; D012335:RNA, Ribosomal / Q000737:chemistry* / Q000378:metabolism*; D012336:RNA, Ribosomal, 16S / Q000737:chemistry / Q000378:metabolism; D012338:RNA, Ribosomal, 23S / Q000737:chemistry / Q000378:metabolism; D016914:Ribonuclease H; D012270:Ribosomes / Q000378:metabolism*; D018271:Signal Recognition Particle / Q000737:chemistry / Q000378:metabolism*; D013891:Thiouridine / Q000737:chemistry
C409968:4.5S RNA; D003432:Cross-Linking Reagents; D029968:Escherichia coli Proteins; C082931:Ffh protein, E coli; D046911:Macromolecular Substances; D012329:RNA, Bacterial; D012335:RNA, Ribosomal; D012336:RNA, Ribosomal, 16S; D012338:RNA, Ribosomal, 23S; D018271:Signal Recognition Particle; D013891:Thiouridine; D016914:Ribonuclease H