Motivation and Aim:
Traditionally, heat-shock proteins are believed to be involved in protein folding traffi cking and rescuing of improperly folded polypeptides. Nevertheless, a number of chaperones have been found out in interaction with macromolecules other then proteins. Some of the molecular chaperones can participate in regulation of mRNA translation. It was described for yeast chaperone Zuotin from Hsp40/DnaJ family [1] and plant chaperone Hsp101from Hsp100/ClpB [2]. We have shown that expression of the reporter gene under control of CUP1 promoter is positively regulated by Hsp104 independently on the level of transcription [3]. It was suggested that the molecular chaperones regulate expression of some particular genes binding with untranslated region (UTR) of the corresponding mRNA.
Methods:
Secondary structure of CUP1 5’-UTR was performed by mfold (version 3.2). Potential binding sites for transcription factors and control elements was found using MatInspector. Searching for translation regulation signals was done using BLASTN 2.2.10. Amino-acid sequence of chaperones that take part in regulation of gene expression were obtained from the GenBank. Sequence alignment and comparative analysis was performed using ClustalW and BioEdit. Protein structure was obtained from Pfam and PDB databases. Experiments were carried out with Saccharomyces cerevisiae strain BY4742 (Invitrogen). A set of reporter constructions was created using methods of molecular cloning and site- directed mutagenesis. Transcription rates were estimated using reverse transcription real time PCR technique with fl uorescent TaqMan probes. Reporter protein translation was monitored using fl uorescence microscopy. Amount of proteins was compared using immunoblotting followed with densitometry.
Results and Conclusion:
Four tandem repeats (CAAU) in 5’-UTR from CUP1 mRNA are essential for translation regulation. We have shown that CUP1 5’-UTR participates in Hsp104 mediated post-transcriptional regulation of gene expression. We have also found the potential domains that involved in Hsp104 – mRNA interaction and proposed the hypotheses of Hsp104 mediated regulation of gene expression in yeast Saccharomyces cerevisiae.
Acknowledgements:
The work was supported by FTP “Academic and teaching staff of innovative Russia” of The Russian Ministry of Education and Science.
Reference:
1. Santanu Raychaudhuri S et al. (2006) Zuotin. A DnaJ molecular chaperone, stimulates cap-independent translation in yeast // Biochem. and Biophys. Res. Com. V.350, P.788-795.
2. Gallie DR et al. (1987) The 5’-leader sequence of tobacco mosaic virus RNA enhances the expression of foreign gene transcripts in vitro and in vivo // Nucl. Acids Res. V.15, P.3257-3273.
3. Rubel AA et al. (2008) Yeast chaperone Hspl04 regulates gene expression on the posttranscriptional level // Mol. Biol. Т.42, №1 С.123-130.