Requirementsfortranslationre-initiationinEscherichiacoli.pptVIP

Further Study Is the 70S ribosome involved in re-initiation? The END! Polymerase Chain Reaction PCR– method for making many copies of a specific segment of DNA, starting with very small amounts (amplifies DNA) DNA to be amplified is mixed with DNA oligonucleotides, Taq polymerase, and primers Mix is heated (break H bonds/sep DNA strands) and cooled (allow DNA primers to anneal) Primers hybridize to ends of gene to be amplified and provide a starting point for Taq P. which synthesizes complementary strands of DNA Go through “thermal cycling” –until enough DNA has been produced Transformation Add transformation solution (ex CaCl2) to tube Place on ice and add E.coli colony Add fragment containing gene(s) of interest (incubate on ice) Heat shock – place tube into a warm heat bath for a little under a minute, place back on ice (about 2 minutes) Add nutrients (LB nutrient broth) and sit at room temp (10 minutes) Transfer to agar plates (these plates contain ampicillin selection and mutants require arabinose inducer) Requirements for translation re-initiation in Escherichia coli:roles of initiator tRNA and initiation factors IF2 and IF3 Molecular Microbiology (2008) 67(5), 1012–1026 Jae-Ho Yoo? and Uttam L. RajBhandary* Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA Presentation by: Liz Gallo Introduction Purpose: investigate requirements for translation re-initiation in E.coli by: constructing a di—cistronic reporter based on the translationally coupled geneV-geneVII pair from M13 phage studying the effects of using mutant initiator tRNA’s studying the effects by modulating IF2 and IF3 activity Key Terms Di-cistronic – translates 2 proteins Polycistronic – translates many proteins Intercistronic region – distance between the stop codon of upstream ORF and the start codon of the downstream ORF Reporter – gene that researchers attach to a regulatory sequence of another gene of interest; often used as an indication