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).

J. Mullet, Aggieland

Young (meristematic) cells w/proplastids

Older cells (etioplasts)

Barley (Hordeum vulgare) 7-10 days old

• Etioplasts lack:

1. Chlorophyll

2. Photosynthetic capacity

3. Major thylakoid membrane proteins

Etioplastlight

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)

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 regulation

CP RNA polymerases

Two 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) 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:1. Endonucleases - cut internally (e.g., between genes),

fairly specific

2. 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:1. proteins recognize the 3'-IR, bind and stop a

processive exonuclease

2. An endonuclease cleaves at the 3’-IR

3. 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) 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 start5'----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

1. 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 translation

Chloroplast tRNAs:Chloroplast translation relies heavily on wobble (or 2 out of 3) pairing between the tRNA anticodon and the mRNA

codon.