Plant Nuclear Gene Expression & Regulation

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.

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

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. 6.33 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).

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)

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!

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: • Upstream element(s) • TATA Box (at approx. –25) • Initiation region (includes the first transcribed nt, +1) • 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: • 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)

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

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.

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: • 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).

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!)

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

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

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 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. 13.25

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

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)

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)

Plant Nuclear Gene Expression & Regulation