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Nucleic Acids Research, 1992, Vol. 20, No. 22 5919-5925
© 1992


MOLECULAR BIOLOGY

Evolutionary conserved nucleotides within the E.coli 4.5S RNA are required for association with P48 in vitro and for optimal function in vivo

Heather Wood*, Joen Luirink+ and David Tollervey

European Molecular Biology Laboratory Postfach 102209, Meyerhofstrasse 1, 6900 Heidelberg, Germany

* To whom correspondence should be addressed

Received September 4, 1992. Revised October 16, 1992. Accepted October 16, 1992.

E.coli 4.5S RNA is homologous to domain IV of eukaryotic SRP7S RNA, the RNA component of the signal recognition particle. The 4.5S RNA is associated in vivo with a 48kD protein (P48), which is homologous to a protein component of the signal recognition particle, SRP54. In addition to secondary structural features, a number of nucleotides are conserved between the 4.5S RNA and domain IV of all other characterised SRP-like RNAs from eubacteria, archaebacteria and eukaryotes. This domain consists of an extended stem-loop structure; conserved nucleotides lie within the terminal loop and within single-stranded regions bulged from the stem immediately preceding the loop. This conserved region is a candidate for the SRP54/P48 binding site. To determine the functional importance of this region within the 4.5S RNA, mutations were introduced into the 4.5S RNA coding sequence. Mutated alleles were tested for their function in vivo and for the ability of the corresponding RNAs to bind P48 in vitro. Single point mutations in conserved nucleotides within the terminal tetranucleotide loop do not affect P48 binding in vitro and produce only slight growth defects. This suggests that the sequence of the loop may be important for the structure of the molecule rather than for specific interactions with P48. On the other hand, nucleotides within the single-stranded regions bulged from the stem were found to be important both for the binding of P48 to the RNA and for optimal function of the RNA in vivo.


+ Present address: Department of Molecular Microbiology, Faculty of Biology, De Boelelaan 1087, 1081 HV Amsterdam, The Netherlands


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