Transcript Nuclear gene expression 1

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.
Likely order of events in producing a
mature mRNA from a pre-mRNA.
Transcription: 3 DNA-Dependent RNA
Polymerases
1. Pol I - synthesizes 45S rRNA precursor, found
in nucleoli (45S18S, 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).
Relative cellular RNA abundance
• Ribosomal RNAs (rRNAs)
• Transfer RNAs (tRNAs)
• Messenger RNAs (mRNAs)
The rest (~3%):
• Signal recognition particle (SRP) RNA
• Small nuclear RNAs (snRNAs)
• Small nucleolar RNAs (snoRNAs)
• Micro RNAs (miRNAs)
~ 90%
~ 5%
~ 2%
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!
TFII – transcription
factors for RNA Pol II
RNAPII – RNA Pol II
Fig. 6.30, Buchanan et al.
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
1.
2.
3.
4.
Many class II promoters lack 3 and 4; a few lack 2.
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)
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)
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
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 proximalpromoter 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.
Assembly of the RNA Pol II
Initiation Complex
= basal factors + RNAP II
TFIIF delivers Pol II
TFIIH PO4ylates the
LS of Pol II, allowing
it to escape the
promoter.
Fig. 7.45, Buchanan et al.
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).
DNA-binding domains
1.
2.
3.
4.
Zinc – containing modules
Homeodomains (conserved amino acid seq.)
bZIP and bHLH motifs
AP2 (mainly in plants)
(not an exhaustive list, just what might be on the test!)
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
3 Models of possible enhancer action.
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
H2 A H3
H2B
H1
H4
H2 B
DNA
Nucleosome core = octamer of histones (2 each of H2A,
H2B, H3, H4) + 2 wraps (145 bp) of DNA
Packing ratio ~5
Histones can be modified (for chromatin remodeling)
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.
Fig. 7.49 Buchanan et al.
In Vivo Studies
• Promoters of active genes are often
deficient in nucleosomes
SV40 virus
minichromosomes
with a nucleosomefree zone at its twin
promoters.
Can also be shown for cellular
genes by DNase I digestion of
chromatin – promoter regions
are hypersensitive to DNase I.
Fig. 13.25
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
Cap Functions
•
Capping also includes methylation of the
ribose (2-OH) on nt #1 and sometimes #2.
•
1.
2.
3.
4.
Cap functions:
Protection from 5 exoribonucleases
Enhances translation in the cytoplasm
Enhances transport from the nucleus
Enhances splicing of the first intron (for
some pre-mRNAs)
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
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)