CHLOROPLASTGENEEXPRESSION-…叶绿体基因的表达—….pptVIP

Translation in Chloroplasts Translation machinery is bacteria-like: Ribosomes: -70S (composed of L (50S) and S (30S) subunits) -contain 23S (L), 16S (S), and 5S (L) rRNAs -each subunit (L and S) contains ~30 proteins Initiation factors: if1, if2, if3 Elongation factors: ef-Tu, ef-Ts, and G Translation is initiated with fmet (formylated Met) Chloroplast polyribosomes will use E. coli soluble factors for elongation and termination phases. How mRNAs selected for translation? Many cp mRNAs contain a Shine-Dalgarno sequence preceding the first codon; it base-pairs to the 3-end of 16S rRNA. S-D start 5----GGAGG-------AUG-----3’ mRNA 3----CCUCC--------5 16S rRNA Start codon (AUG) very important for starting translation at right codon. Can translate internal ORFs of a polycistronic transcript. In vitro translation w/chloroplast extract: Hirose and Sugiura, 1996. EMBO J. 15, 1687–1695. mRNA recognition/binding using the Shine-Dalgarno sequence in plastid mRNAs Fig. 9.17 Differences with bacteria Many chloroplast mRNAs have relatively long (~ 300 nt) 5 untranslated regions (UTR) that bind proteins. Many chloroplast mRNAs don’t have a S-D sequence, and in 1 case, it suppresses translation (Sugiura lab). Must be another initiation mechanism Scanning ? Some of the proteins that bind the 5’ UTRs of mRNAs promote translation Chloroplast tRNAs: Chloroplast translation relies heavily on wobble (or 2 out of 3) pairing between the tRNA anticodon and the mRNA codon. * * * Hybridization with a psbC probe, lanes 6-9 are after 16, 36, 72 or 108 hr of light * * * * * * * * * * * * * * * (a) 5’ processing can be either by a 5’ to 3’ exo that is stopped by a bound protein or by an endonuclease (b) Endo cleaves internally, fragments degraded by exonucleases or a 5’ to 3’ processive endonuclease Not shown in the photo – Fragments can get polyadenylated and get degraded quickly by polynucleotide phosphorylase, which can also put on the polyA-ta