The efficiency of a cis-cleaving ribozyme in an mRNA coding region is influenced by the translating ribosome in vivo.
A cis -cleaving hammerhead ribozyme (Rz) expression system (3A'-Rz) in Escherichia coli has been constructed that can be used to study the involvement of factors that affect ribozyme cleavage in vivo . The ribozyme sequence is placed in the coding region of 3A' mRNA, which is expressed from a semi-synthetic translation assay gene. The size and the 5'-end sequences of the 3' cleavage fragments were determined and the efficiencies of different Rz variants were measured by quantitative primer extension. It is shown that one of the semi-active constructs (3A'-RzIII) can be used as an indicator for ribosomes that read through or terminate at a stop codon upstream of the Rz hammerhead sequence in the mRNA. Readthrough of the stop codon in an uncleaved mRNA gives a full length 3A' protein. Termination at the stop codon upstream of the ribozyme sequence gives a shortened termination product. However, the mRNA fragment that should arise as a result of the auto-cleavage does not give rise to any detectable corresponding truncated protein. Besides studies on translating ribosomes, the 3A'-Rz system can be used to isolate mutant strains that are changed in ribozyme activity either from internal base alterations, or changed interacting host factors.
Zhang
S
S
Department of Microbiology, Stockholm University, S-106 91 Stockholm, Sweden.
Stancek
M
M
Isaksson
L A
LA
D001483:Base Sequence; D018388:Codon, Terminator; D004926:Escherichia coli / Q000235:genetics; D017930:Genes, Reporter / Q000235:genetics; D005813:Genes, Synthetic / Q000235:genetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D014176:Protein Biosynthesis; D016337:RNA, Catalytic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism*; D013205:Staphylococcal Protein A / Q000096:biosynthesis / Q000235:genetics
D018388:Codon, Terminator; D016337:RNA, Catalytic; D012333:RNA, Messenger; D013205:Staphylococcal Protein A