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). 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) .

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 – 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.

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!) 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.

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. (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.

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) 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: 7MeGpppX1pX2.... (X1 and X2 are transcribed ribonucleotides).

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) 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