Lecture 12

Lecture 12 - PLB 113 - Lecture 12 II. Chloroplast...

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PLB 113 - Lecture 12 II. Chloroplast Biogenesis F. Protein Transport G. Transit Peptide Receptor * Next Discussion Session: 2060 SciLab ----------------------------------------------------------------------------------------------------------------------------- - Review: In vitro import of the precursor of the small subunit of Rubsico When prSS + chloroplast + ATP + Light = - some of the SS will be in the chloroplast. = as matSS - some of the SS won't get into the chloroplast. = as prSS * import of prSS associates with the lost of TP (two bands on the 2nd column) To tell which form is inside/outside of chloroplast: - add protease (degrade protein but cannot get into chloroplast) - proteins outside the chloroplast will be degraded = prSS are degraded - proteins inside the chloroplast will be protected = matSS are remaining (single band on the last column) In Vitro Import of mature small subunit of Rubisco matSS = no TP = none of them get into chloroplast - with protease = proteins outside the chloroplast are degraded = no band Other way to analyze it, instead of adding protease, use " reisolation " = same results = only see the proteins present in the chloroplast Question of the Day: What's the origin of transit peptide? 1) TP arose from accumulation of a set of mutations on 5' untranlated region of the gene which
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generates the additional coding sequences at the amino terminus. 2) TP were created by DNA rearrangement that added the additional block of amino acids to the amino terminus of the original protein. * genes coming from chloroplast genome have been inserted into nucleus genome * what are the chances that you can have either mutation or rearrangement? Idea from an Experiment Done on Mitochondria : - take a gene from mitochondrial protein: cox gene, which involves the mitochondrial respiration - eliminate TP from cox gene - take random fragments from the genome of Ecoli and from the genome of mouse DHFR gene - fuse the random fragments with COX gene to make "chimeric gene" - transform chimeric gene into yeast mutant (cox-) [mutation on the endogenous cox gene] * this mutant cannot grow unless it has a wild type cox gene, which encodes a protein that's present in mitochondria. * in order to get back to a "living" yeast, cox protein has to be present in the mitochondria * the only source of the cox protein is coming from the chimeric gene Logic: asking how often can a random fragment of DNA serve as transit peptide? The study found: Ecoli > 2.7 % of the clones rescue the mutation = 2.7% of the random fragments inserted next to the cox gene would serve as a TP which was sufficient to get cox protein into the mitochondria. Mouse > 5% of the clones rescue the mutation = high frequency = taking a random sequence inserted next to the cox gene serve as TP Infer that: Through evolution, chloroplast genes have been inserted randomly into the genome. The sequence next to the protein that get inserted somehow is about to serve as a TP for that protein.
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Lecture 12 - PLB 113 - Lecture 12 II. Chloroplast...

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