Escherichia coli initiation factor 3 protein binding to 30S ribosomal subunits alters the accessibility of nucleotides within the conserved central region of 16S rRNA.
Translational initiation factor 3 (IF3) is an RNA helix destabilizing protein which interacts with strongly conserved sequences in 16S rRNA, one at the 3' terminus and one in the central domain. It was therefore of interest to identify particular residues whose exposure changes upon IF3 binding. Chemical and enzymatic probing of central domain nucleotides of 16S rRNA in 30S ribosomal subunits was carried out in the presence and absence of IF3. Bases were probed with dimethyl sulfate (DMS), at A(N-1), C(N-3), and G(N-7), and with N-cyclohexyl-N'-[2-(N-methyl-4-morpholinio)ethyl] carbodiimide p-toluenesulfonate (CMCT), at G(N-1) and U(N-3). RNase T1 and nuclease S1 were used to probe unpaired nucleotides, and RNase V1 was used to monitor base-paired or stacked nucleotides. 30S subunits in physiological buffers were probed in the presence and absence of IF3. The sites of cleavage and modification were detected by primer extension. IF3 binding to 30S subunits was found to reduce the chemical reactivity and enzymatic accessibility of some sites and to enhance attack at other sites in the conserved central domain of 16S rRNA, residues 690-850. IF3 decreased CMCT attack at U701 and U793 and V1 attack at G722, G737, and C764; IF3 enhanced DMS attack at A814 and V1 attack at U697, G833, G847, and G849. Many of these central domain sites are strongly conserved and with the conserved 3'-terminal site define a binding domain for IF3 which correlates with a predicted cleft in two independent models of the 30S ribosomal subunit.
Muralikrishna
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Department of Chemistry, University of South Florida, Tampa 33620.
Wickstrom
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D001426:Bacterial Proteins / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D002352:Carrier Proteins / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics*; D006868:Hydrolysis; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010448:Peptide Initiation Factors / Q000235:genetics* / Q000378:metabolism*; D039665:Prokaryotic Initiation Factor-3; D012335:RNA, Ribosomal / Q000378:metabolism*; D012336:RNA, Ribosomal, 16S / Q000378:metabolism*; D016601:RNA-Binding Proteins; D006163:Ribonuclease T1; D012269:Ribosomal Proteins / Q000302:isolation & purification / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism*; D015719:Single-Strand Specific DNA and RNA Endonucleases
D001426:Bacterial Proteins; D002352:Carrier Proteins; D010448:Peptide Initiation Factors; D039665:Prokaryotic Initiation Factor-3; D012335:RNA, Ribosomal; D012336:RNA, Ribosomal, 16S; D016601:RNA-Binding Proteins; D012269:Ribosomal Proteins; C015494:ribosomal protein S1; D006163:Ribonuclease T1; D015719:Single-Strand Specific DNA and RNA Endonucleases