plant nuclear gene expression & regulation
DESCRIPTION
Plant Nuclear Gene Expression & Regulation. A lot of steps to regulate: Transcription * Capping 3' maturation, cleavage & polyadenylation Splicing * Transport to Cytoplasm Stabilization/Destabilization of mRNA * Translation *. * have the most regulation. - PowerPoint PPT PresentationTRANSCRIPT
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Plant Nuclear Gene Expression & Regulation
A lot of steps to regulate:
1. Transcription*
2. Capping
3. 3' maturation, cleavage & polyadenylation
4. Splicing*
5. Transport to Cytoplasm
6. Stabilization/Destabilization of mRNA*
7. Translation*
* have the most regulation.
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Likely order of events in producing a mature mRNA from a pre-mRNA.
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Transcription: 3 DNA-Dependent RNA Polymerases
1. Pol I - synthesizes 45S rRNA precursor, found in nucleoli (45S18S, 28S, 5.8S rRNAs)
[S refers to rate of sedimentation (Fig. 6.33 in Buchanan), approx. equivalent to size of macromolecule]
2. Pol II - synthesizes mRNA precursors, some snRNAs
3. Pol III- synthesizes 5S rRNAs, tRNAs, small nuclear RNAs (snRNAs)
All 3 polymerases are multi-subunit; have some large, unique subunits; and 5 small, shared subunits (at least in yeast).
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Relative cellular RNA abundance
• Ribosomal RNAs (rRNAs) ~ 90%• Transfer RNAs (tRNAs) ~ 5%• Messenger RNAs (mRNAs) ~ 2%
The rest (~3%):• Signal recognition particle (SRP) RNA • Small nuclear RNAs (snRNAs)• Small nucleolar RNAs (snoRNAs)• Micro RNAs (miRNAs)
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RNA Polymerase II
1. 2 large subunits have regions of homology with ß and ß’ subunits of E. coli RNAP.
2. Largest subunit is phosphorylated on its COOH-terminal domain (CTD)– Phosphor. needed for transition from initiation
elongation– CTD also interacts with other proteins
3. Does not bind DNA by itself, requires other proteins to bind promoter first!
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Fig. 6.30, Buchanan et al.
TFII – transcription factors for RNA Pol II
RNAPII – RNA Pol II
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RNAP II Promoters
• Class-II promoters have 4 components:1. Upstream element(s)2. TATA Box (at approx. –25)3. Initiation region (includes the first transcribed nt, +1)
4. Downstream element
Many class II promoters lack 3 and 4; a few lack 2.
1. 2. 3. 4.
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TATA Box of Class II Promoters
• TATA box = TATAAAA
• Defines where transcription starts
• Also required for efficient transcription for some promoters
• Bound by TBP – TATA box binding protein (in complexes like TFIID)
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Upstream elements: Class II promoters
Found in many class II promoters:
1. GC boxes (GGGCGG and CCGCCCC)– Stimulate transcription in either orientation– May be multiple copies– Must be close to TATA box
2. CCAAT box
– Stimulates transcription– Binds CTF (Cat-box transcription factor)
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Enhancers and Silencers
1. Enhancers stimulate transcription, while Silencers inhibit.
2. Orientation-independent– Flip 180 degrees, still work
3. Position-independent (mostly) – Can work at a distance from promoter core– Enhancers have been found all over
4. Bind regulatory transcription factors
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Transcription factors for Class II promoters
1. Basal factors: required for initiation at most promoters; interact with TATA box.
2. Upstream factors: bind common (consensus) elements upstream of TATA, including proximal-promoter elements (e.g., CCAAT box); increase efficiency of initiation.
3. Inducible (regulated) factors: work like upstream factors but are regulatory (produced or active only at specific times/tissues); interact with enhancers or silencers.
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Assembly of the RNA Pol II Initiation Complex= basal factors + RNAP II
Fig. 7.45, Buchanan et al.
TFIIF delivers Pol II
TFIIH PO4ylates the LS of Pol II, allowing it to escape the promoter.
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Eukaryotic Transcription Factors: Structure
• Mostly about factors that bind USEs:
1. Modular structure:– DNA-binding domain– Transcription-activating domain
2. Can have > 1 of each type of module 3. Many factors also have a dimerization
domain (some can form heterodimers).
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DNA-binding domains
1. Zinc – containing modules2. Homeodomains (conserved amino acid seq.)
3. bZIP and bHLH motifs
4. AP2 (mainly in plants)
(not an exhaustive list, just what might be on the test!)
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Activation from a Distance: Enhancers
• 3 possible models
Factor binding induces:
1. Supercoiling of the promoter DNA
2. Sliding of the complex to the promoter
3. Looping out of DNA between enhancer and promoter
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3 Models of possible enhancer action.
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Chromatin Modification
• Transcription can also be regulated by modifying chromatin (histones); highly transcribed
genes have less condensed chromatin.
• Basic unit of chromatin is the nucleosome:
1. 4 different histones in the core (H2a, H2b, H3, H4 x 2 = octamer)
2. 146 bp of DNA wrapped around core
3. Histone H1 on outside
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H2B
H4
H3
H1
H2B
H2A
DNA
Packing ratio ~5
Nucleosome core = octamer of histones (2 each of H2A, H2B, H3, H4) + 2 wraps (145 bp) of DNA
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Fig. 7.49 Buchanan et al.
Histone acetylation (right) causes localized unpacking of nucleosomes, which enhances factor binding to DNA.
De-acetylated histones (left) bind DNA more strongly, and the nucleosomes condense into a solenoid; this inhibits factor binding to DNA targets.
Histones can be modified (for chromatin remodeling)
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In Vivo Studies • Promoters of active genes are often
deficient in nucleosomes
SV40 virus minichromosomes with a nucleosome-free zone at its twin promoters.
Fig. 13.25
Can also be shown for cellular genes by DNase I digestion of chromatin – promoter regions are hypersensitive to DNase I.
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Post-Transcriptional Processes
1. Capping
2. 3’ end formation (not much regulation of the above
steps)
3. Splicing – alternative splicing
4. Translation – regulate initiation step
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Cap Functions
• Capping also includes methylation of the ribose (2-OH) on nt #1 and sometimes #2.
• Cap functions:
1. Protection from 5 exoribonucleases
2. Enhances translation in the cytoplasm
3. Enhances transport from the nucleus
4. Enhances splicing of the first intron (for some pre-mRNAs)
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3’ end Processing & Polyadenylation Mechanism
• Transcription extends beyond mRNA end
• Transcript is cut at 3’ end of what will become the mRNA
• PolyA Polymerase adds ~250 As to 3’ end
• “Extra” RNA degraded
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3' End Formation
CIS (elements)• AAUAA is the key signal in higher plants, its
found ~20 nt from the polyA-tail.– Other sequences 5' to the AAUAA also
important.
TRANS (factors)• 3' end formation requires at least:
– an endonuclease & recognition factors– a poly(A) polymerase (PAP)– a poly A-binding protein (PAB)