Investigation On The Roles Of The Non-coding Regions On The Expression Of Arabidopsis Ribosomal Protein L36B Gene

Ribosome is the place where protein synthesis takes place. The rate of ribosome synthesis is economically coordinated with the physiological states of cells. Expression of the components for ribosome assembly, rRNAs and ribosomal proteins, are also exquisitely regulated so that stoichiometric amounts of rRNAs and ribosomal proteins can accumulate. Gene expression can be regulated to different extents by non-coding regions, such as promoter, intron, and untranslated regions (UTRs). Results generated in our laboratory have idetified two highly conserved motifs (motif1, motif2) present in more than 95% of Arabidopsis ribosomal protein gene promoters at -80 or -50 positions from the transcriptional start sites. As also seen in Yeast and Human ribosomal protein genes, in almost all Arabidopsis ribosomal protein genes, there is a structurally conserved intron very close to the translational initiation codon. To investigate the mechanism underlying the coordinated expression of RP genes in plant, we chose Arabidopsis RPL36B gene as an example for detailed analysis of the roles of different non-coding regions. We have also selected, from Arabidopsis genome database, actin2 promoter, which lacks both motif1 and motif2 as well as TATA box in its promoter, and fructose bisphosphate aldolase (FBA) 5’UTR sequence, which has a similar length as the 5’UTR of RPL36B in addition to having a similar promoter structure as actin2, to be used in the preparation of experimental constructs. We have made all together six constructs, the wild type RPL36B gene; a mutant in which the 5’ promoter was replaced by actin2 promoter; a mutant in which the first intron was deleted; a mutant in which the 5’ promoter was replaced by actin2 promoter and the first intron was also deleted; a mutant in which the 5’ UTR was replaced by the one from FBA; and a mutant in which the first intron was moved to a farther position from the translational start site. In order to more precisely mimic the environment in plant cells, these constructs were transformed through Agrobacterium tumefaciens GV3101 into carrot callus culture and tobacco BY2 suspension cells for examination of their regulated expression. Integration of these experimental constructs into plant genome was confirmed by GUS staining. We are planning to perform Northern analysis and Western detection have been taken to examine mRNA expression from these transgenes and the abundance of the Arabidopsis RPL36 proteins by exploiting the HA tag present in the transgenes. Sucrose gradient fractionation combined with Northern analysis will be performed to examine the translational efficiencies of the mRNAs derived from these constructs under different physiological conditions, with or without plant hormone 2, 4-D, to search for the non-coding regions important in the coordinated regulation of ribosomal protein genes in plant.