Changes in the 5'-untranslated region of the rbcL gene accelerate transcript degradation more than 50-fold in the chloroplast of Chlamydomonas reinhardtii.
Using uidA (beta-glucuronidase; GUS) reporter gene constructs, the 5'-untranslated region (UTR) of the Chlamydomonas chloroplast rbcL gene was screened by deletion and mutational analysis for the presence of a promoter element that previous studies implied to reside within the first 63 base pairs of the UTR. Deleting a large segment of the rbcL 5'UTR in a 3'-->5' direction to position +36, changing the remaining 36 base pairs at the 5' end of the UTR, and increasing by five base pairs the distance between the rbcL 5'UTR and the basic promoter element located at position -10 did not abolish transcription from the basic rbcL promoter. It is concluded that the apparent loss of transcriptional activity found in earlier studies after deletion of sequences downstream of the transcription initiation site is due to the synthesis of very unstable transcripts that escape detection by Northern analysis and in vivo transcription assays. Chimeric rbcL:GUS transcripts containing changes in the beginning of the 5'UTR that affect RNA secondary structure are estimated to be at least 50 times less stable than rbcL:GUS transcripts containing the non-modified rbcL 5'UTR sequence.
Salvador
Maria Luisa
ML
Department of Biochemistry and Molecular Biology, University of Valencia, Dr. Moliner 50, 46100, Burjassot, Valencia, Spain.
Suay
Loreto
L
Anthonisen
Inger Lill
IL
Klein
Uwe
U
D020121:5' Untranslated Regions / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D002678:Chimera / Q000235:genetics; D016825:Chlamydomonas reinhardtii / Q000235:genetics*; D002736:Chloroplasts / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000235:genetics; D020871:RNA Stability / Q000235:genetics*; D012273:Ribulose-Bisphosphate Carboxylase / Q000235:genetics*; D017384:Sequence Deletion / Q000235:genetics; D014158:Transcription, Genetic / Q000235:genetics
D020121:5' Untranslated Regions; C101625:RbcL protein, plastid; D012273:Ribulose-Bisphosphate Carboxylase