CHLOROPLAST GENE EXPRESSION Transcription RNA processing (splicing, cleavages, modification) Translation Regulation Dependence on nuclear genes

TRANSCRIPTION 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).

Hybridization with a psbC probe, lanes 6-9 are after 16, 36, 72 or 108 hr of light J. Mullet, Aggieland

Barley (Hordeum vulgare) 7-10 days old Older cells (etioplasts) Young (meristematic) cells w/proplastids

Etioplasts lack: Chlorophyll Photosynthetic capacity Major thylakoid membrane proteins light Etioplast Chloroplast

How 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) Distance between -10 and -35 regions critical " " -10 and start (+1) less critical Much variablility in the consensus sequence 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 regulation

CP RNA polymerases Two main forms in vascular plants: E. coli or eubacterial-like polymerase (also called PEP, plastid-encoded polymerase) Phage-like or NEP (nuclear-encoded polymerase) polymerase

E. 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 rifampicin

Fig. 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 genes

CP pre-mRNA PROCESSING Most, if not all primary transcripts are processed by cleavage(s) or splicing or both CP mRNAs are not polyadenylated, and are not "capped" (cap= 7methylguanosine). Nucleolytic Cleavages: Endonucleases - cut internally (e.g., between genes), fairly specific Exonucleases - trim at 3' or 5'-ends, processive, less specific

Inverted repeats in cpRNA processing Inverted repeats occur at 3'-end of most cp protein-encoding genes. - processing sites, determine the 3'-end of mRNAs mechanisms: proteins recognize the 3'-IR, bind and stop a processive exonuclease An endonuclease cleaves at the 3’-IR Combination of the two above

3’- end processing and stabilization of chloroplast mRNAs D. Stern, Cornell

Pathways of Cp pre-mRNA Processing & Degradation in Chlamydomonas (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-tail (reversible), PAP has not been proven in chloroplast (a) and (b) may use some of the same enzymes D. Stern, Cornell

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. The chloroplast encodes only ~30-33 different tRNAs (some encode many less), but that’s enough to read 61 codons if we assume wobble base pairing in the third position of the codon (i.e., only the first 2 nt in the codon need form Watson-Crick pairs with the anticodon)