Tagging ribosomal protein S7 allows rapid identification of mutants defective in assembly and function of 30 S subunits.
Ribosomal protein S7 nucleates folding of the 16 S rRNA 3' major domain, which ultimately forms the head of the 30 S ribosomal subunit. Recent crystal structures indicate that S7 lies on the interface side of the 30 S subunit, near the tRNA binding sites of the ribosome. To map the functional surface of S7, we have tagged the protein with a Protein Kinase A recognition site and engineered alanine substitutions that target each exposed, conserved residue. We have also deleted conserved features of S7, using its structure to guide our design. By radiolabeling the tag sequence using Protein Kinase A, we are able to track the partitioning of each mutant protein into 30 S, 70 S, and polyribosome fractions in vivo. Overexpression of S7 confers a growth defect, and we observe a striking correlation between this phenotype and proficiency in 30 S subunit assembly among our collection of mutants. We find that the side chain of K35 is required for efficient assembly of S7 into 30 S subunits in vivo, whereas those of at least 17 other conserved exposed residues are not required. In addition, an S7 derivative lacking the N-terminal 17 residues causes ribosomes to accumulate on mRNA to abnormally high levels, indicating that our approach can yield interesting mutant ribosomes.
Fredrick
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K
Center for Molecular Biology of RNA, University of California, Santa Cruz, CA 95064, USA.
Dunny
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GM
Noller
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HF
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D002455:Cell Division; D017124:Conserved Sequence / Q000235:genetics; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry / Q000166:cytology / Q000235:genetics; D015870:Gene Expression; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics*; D011132:Polyribosomes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D014176:Protein Biosynthesis; D017433:Protein Structure, Secondary; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012269:Ribosomal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012270:Ribosomes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D017384:Sequence Deletion / Q000235:genetics; D013329:Structure-Activity Relationship
D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D012269:Ribosomal Proteins; C037442:ribosomal protein S7; D017868:Cyclic AMP-Dependent Protein Kinases