Abstract
Chlamydomonas reinhardtii is a widely used model organism to study chloroplast gene expression and photosynthetic processes. In this project we worked to identify a novel trans-acting factor that is thought to contribute to the stability of rbcL mRNA. These types of factors are thought to bind to specific cis-sequence elements and protect the transcript from nuclease degradation.
Experimental studies have proven that the 5‟untranslated region of Chlamydomonas rbcL mRNA plays a key role in the regulation of rbcL transcript stability. Changing nucleotides in the 5‟UTR renders rbcL transcripts unstable, and it is thought that sequence elements within the transcripts recruit and bind proteins that functions as trans-acting factors.
In this project we cloned a protein (RB60) that is thought to have RNA-binding capabilities to Chlamydomonas rbcL 5‟UTR. The protein was cloned into a transformation vector and introduced into E.coli.
UV cross-linking experiments showed that there is no conclusive evidence that RB60 can bind to Chlamydomonas rbcL 5‟UTR. High concentration of RB60 protein yielded low affinity binding to rbcL 5‟UTR sequences in vitro. Control experiments with E.coli extract without RB60 construct identified a bacterial protein that has high affinity for 5‟UTR sequences in vitro.