Escherichia coli proteins, including ribosomal protein S12, facilitate in vitro splicing of phage T4 introns by acting as RNA chaperones.
To address the effect of host proteins on the self-splicing properties of the group I introns of bacteriophage T4, we have purified an activity from Escherichia coli extracts that facilitates both trans- and cis-splicing of the T4 introns in vitro. The activity is attributable to a number of proteins, several of which are ribosomal proteins. Although these proteins have variable abilities to stimulate splicing, ribosomal protein S12 is the most effective. The activity mitigates the negative effects on splicing of the large internal open reading frames (ORFs) common to the T4 introns. In contrast to proteins shown previously to facilitate group I splicing, S12 does not bind strongly or specifically to the intron. Rather, S12 binds RNA with broad specificity and can also facilitate the action of a hammerhead ribozyme. Addition of S12 to unreactive trans-splicing precursors promoted splicing, suggesting that S12 can resolve misfolded RNAs. Furthermore, incubation with S12 followed by its proteolytic removal prior to the initiation of the splicing reaction still resulted in splicing enhancement. These results suggest that this protein facilitates splicing by acting as an RNA chaperone, promoting the assembly of the catalytically active tertiary structure of ribozymes.
Molecular Genetics Program, Wadsworth Center, New York State Department of Health, Albany 12201-0509.
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D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism*; D017122:Bacteriophage T4 / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D007438:Introns; D007700:Kinetics; D018832:Molecular Chaperones / Q000378:metabolism*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D016366:Open Reading Frames; D012322:RNA Precursors / Q000378:metabolism; D012326:RNA Splicing; D016337:RNA, Catalytic / Q000378:metabolism; D012367:RNA, Viral / Q000096:biosynthesis*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D012269:Ribosomal Proteins / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D018832:Molecular Chaperones; D012322:RNA Precursors; D016337:RNA, Catalytic; D012367:RNA, Viral; D016601:RNA-Binding Proteins; D012269:Ribosomal Proteins; C034212:ribosomal protein S12; D006160:Guanosine Triphosphate