CHLOROPLAST GENE EXPRESSIONTRANSCRIPTIONSlide 3Slide 4Slide 5How many promoters in cpDNA?Slide 7Cp PromotersControl of Cp transcriptionCP RNA polymerasesE. coli-like (PEP) polymeraseSlide 12Phage-like (NEP) polymeraseSlide 14CP pre-mRNA PROCESSINGInverted repeats in cpRNA processingSlide 17Slide 18Translation in ChloroplastsHow mRNAs selected for translation?mRNA recognition/binding using the Shine-Dalgarno sequence in plastid mRNAsDifferences with bacteriaSlide 23CHLOROPLAST GENE EXPRESSION•Transcription•RNA processing (splicing, cleavages, modification)•Translation•Regulation•Dependence on nuclear genesTRANSCRIPTION•Many, but not all, cp genes are arranged in operon-like units and co-transcribed–e.g., psbD-psbC gene cluster (see next slide)–A unique feature of psbD-psbC gene transcription: a different (closer) promoter is used in the light called the light-responsive promoter (LRP).J. Mullet, AggielandYoung (meristematic) cells w/proplastidsOlder cells (etioplasts)Barley (Hordeum vulgare) 7-10 days old•Etioplasts lack:1. Chlorophyll2. Photosynthetic capacity3. Major thylakoid membrane proteinsEtioplastlightChloroplastHow many promoters in cpDNA? •~30 transcription units (promoters) in higher plant cp DNA–determined experimentally by capping of cp RNA with guanylyl transferase and radioactive GT32P, and hybridization to cpDNA fragments.–The transferase attaches GMP to the 5’ end of RNAs that have 2 or 3 phosphates•Only primary transcription products have > 1 phosphate at the 5’ end of the RNA.A "transcription unit" is determined by the position of the promoter (5') and terminator (3') signals.Terminators not clearly defined, but tRNA genes seem to be good transcription terminators in chloroplasts.Cp Promoters•Most resemble the major E. coli σ70 (or -10,-35) promoter; the consensus sequence is:-35 -10 +1 TTGACA-------TATAAT------AAC--- (DNA) 5’ UUG… (RNA)1. Distance between -10 and -35 regions critical2. " " -10 and start (+1) less critical3. Much variablility in the consensus sequence4. no -10, -35 for some cp genes (i.e. not always required, at least 1 other type of promoter)Control of Cp transcription•Transcription rate important :– mainly controlled at initiation step– determined in part by "promoter strength“– also modulated for some genes (psbD) by upstream sequences that bind regulatory proteins •Some genes have "alternative promoters"(e.g., psbD – psbC) - also provides for regulationCP RNA polymerasesTwo main forms in vascular plants: 1. E. coli or eubacterial-like polymerase (also called PEP, plastid-encoded polymerase)2. Phage-like or NEP (nuclear-encoded polymerase) polymeraseE. coli-like (PEP) polymerase•composed of Core + Sigma factor–Core = 4 subunits, α2 ββ' •α is encoded by the rpoA gene•β is encoded by the rpoB gene•β' is encoded by the rpoC1 and rpoC2 genes–Sigma factor needed to initiate transcription at the bacterial promoter (recognizes -10,-35 regions) •Nuclear encoded, family of 6 genes in Arabidopsis •Inhibited by rifampicinFig. 6.31 in Buchanan et al.Phage-like (NEP) polymerase•Catalytic subunit is similar to the 1-subunit phage (e.g., T7) and mitochondrial RNA polymerases•Nuclear gene•Enzyme insensitive to rifampicin•Promoter is usually a single region of 7-10 bp (YRTA core), but other sequences stimulate •Evolution –Viral Origin?–Mitochondrial origin?–When did it get into plants?Why two chloroplast RNA polymerases?NEP is more important early in plastid development when plastid transcription (and translation) is relatively low.- transcribes rRNA, rpo and other genetic functions genes (GFG)PEP is more important in mature chloroplasts.- transcribes some GFG genes, but strongly transcribes photosynthesis genesCP pre-mRNA PROCESSINGMost, if not all primary transcripts are processed by cleavage(s) or splicing or bothCP mRNAs are not polyadenylated, and are not "capped" (cap= 7methylguanosine).•Nucleolytic Cleavages:1. Endonucleases - cut internally (e.g., between genes), fairly specific2. Exonucleases - trim at 3' or 5'-ends, processive, less specificInverted repeats in cpRNA processingInverted repeats occur at 3'-end of most cp protein-encoding genes.- processing sites, determine the 3'-end of mRNAs -mechanisms:1. proteins recognize the 3'-IR, bind and stop a processive exonuclease2. An endonuclease cleaves at the 3’-IR 3. Combination of the two above3’- end processing and stabilization of chloroplast mRNAsD. Stern, CornellPathways of Cp pre-mRNA Processing & Degradation in Chlamydomonas(a) and (b) may use some of the same enzymesD. Stern, CornellTranslation in ChloroplastsTranslation 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 start5'----GGAGG-------AUG-----3’ mRNA3'----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 mRNAsFig. 9.17Differences with bacteria1. Many chloroplast mRNAs have relatively long (~ 300 nt) 5' untranslated regions (UTR) that bind proteins.2. Many chloroplast mRNAs don’t have a S-D sequence, and in 1 case, it suppresses translation (Sugiura lab).3. Must be another initiation mechanism•Scanning ?•Some of the proteins that bind the 5’ UTRs of mRNAs promote translationChloroplast tRNAs:Chloroplast translation relies heavily on wobble (or 2 out of 3) pairing between the tRNA anticodon and the mRNA