1. 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.

2. Likely order of events in producing a mature mRNA from a pre-mRNA.

3. Transcription: 3 DNA-Dependent RNA Polymerases
Pol I - synthesizes 45S rRNA precursor, found in nucleoli (45S18S, 28S, 5.8S rRNAs)
[S refers to rate of sedimentation (Fig in Buchanan), approx. equivalent to size of macromolecule]
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).
snRNA – small nuclear RNA (e.g., U1, U2). Plants have a 4th RNA Polymerase, Pol IV. its an RNA-dependent RNA Polymerase (also called RdR or RdRP) .

4. 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)

5. RNA Polymerase II
2 large subunits have regions of homology with ß and ß’ subunits of E. coli RNAP.
Largest subunit is phosphorylated on its COOH-terminal domain (CTD)
Phosphor. needed for transition from initiation  elongation
CTD also interacts with other proteins
Does not bind DNA by itself, requires other proteins to bind promoter first!

6. TFII – transcription factors for RNA Pol II
RNAPII – RNA Pol II
Fig. 6.30, Buchanan et al.

7. RNAP II Promoters Class-II promoters have 4 components:
Upstream element(s)
TATA Box (at approx. –25)
Initiation region (includes the first transcribed nt, +1)
Downstream element
Many class II promoters lack 3 and 4; a few lack 2.

8. 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)

9. Upstream elements: Class II promoters
Found in many class II promoters:
GC boxes (GGGCGG and CCGCCCC)
Stimulate transcription in either orientation
May be multiple copies
Must be close to TATA box
CCAAT box
Stimulates transcription
Binds CTF (Cat-box transcription factor)

10. Enhancers and Silencers
Enhancers stimulate transcription, while Silencers inhibit.
Orientation-independent
Flip 180 degrees, still work
Position-independent (mostly)
Can work at a distance from promoter core
Enhancers have been found all over
Bind regulatory transcription factors

11. Transcription factors for Class II promoters
Basal factors: required for initiation at most promoters; interact with TATA box.
Upstream factors: bind common (consensus) elements upstream of TATA, including proximal- promoter elements (e.g., CCAAT box); increase efficiency of initiation.
Inducible (regulated) factors: work like upstream factors but are regulatory (produced or active only at specific times/tissues); interact with enhancers or silencers.

12. Assembly of the RNA Pol II Initiation Complex
= basal factors + RNAP II
TFIIF delivers Pol II
TFIIH – has kinase activity, phosphorylates large subunit of Pol II, needed for Pol II to escape promoter and continue the elongation phase.
TFIIH PO4ylates the LS of Pol II, allowing it to escape the promoter.
Fig. 7.45, Buchanan et al.

13. Eukaryotic Transcription Factors: Structure
Mostly about factors that bind USEs:
Modular structure:
DNA-binding domain
Transcription-activating domain
Can have > 1 of each type of module
Many factors also have a dimerization domain (some can form heterodimers).

14. DNA-binding domains 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!)
1. Zinc fingers (several types) – coordinate Zn2+ with Cys or His, stabilize protein structure
2. Conserved aa sequence (between plants and animals); bind to similar DNA sequences also
3. bZIP – basic region (binds DNA) and a leucine zipper which dimerizes the protein; can form hetero- or homo- dimers
bHLH- basic region, as with bZip, plus a helix-loop-helix motif (dimerize)
4. AP2 – DBDs similar to the DNA-binding domain in the apetala 2 gene (plant transcription factor); ap2 also has an H-N-H motif like the H-N-H homing endonucleases.

15. Activation from a Distance: Enhancers
3 possible models
Factor binding induces:
Supercoiling of the promoter DNA
Sliding of the complex to the promoter
Looping out of DNA between enhancer and promoter

16. 3 Models of possible enhancer action.
(A) Supercoiling model could go in reverse of the direction shown; a factor could cause relaxation. Recently, a topoisomerase has been found to be a transcription factor, induces a DSB that relaxes supercoiling.
(B) DNA-binding proteins are known to slide on DNA.
(C) Good evidence for looping for some factors.
3 Models of possible enhancer action.

17. 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:
4 different histones in the core (H2a, H2b, H3, H4 x 2 = octamer)
146 bp of DNA wrapped around core
Histone H1 on outside

18. Nucleosome core = octamer of histones (2 each of H2A, H2B, H3, H4) + 2 wraps (145 bp) of DNA
Packing ratio ~5

19. 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 Buchanan et al.

20. In Vivo Studies
Promoters of active genes are often deficient in nucleosomes
SV40 virus minichromosomes with a nucleosome-free 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

21. Post-Transcriptional Processes
Capping
3’ end formation (not much regulation of the above steps)
Splicing – alternative splicing
Translation – regulate initiation step

22. Cap Functions
Capping also includes methylation of the ribose (2-OH) on nt #1 and sometimes #2.
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)
Basic capping is the attachment of 7Me-GTP to the 5’ end of a nascent mRNA with a 5’ to 5’ phospho-ester linkage, so it looks like this:
7MeGpppX1pX (X1 and X2 are transcribed ribonucleotides).

23. 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

24. 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)
Endonuclease – probably part of a complex with recognition factors that bind to the AAUAA (and other sequences)
Poly A polymerase – does not need a template, probably limited to ~200 bp of processivity
Poly A binding protein - needed for phase 2 (synthesizing long tail of ~ 200 bp) in mammals