Plant Nuclear Gene Expression & RegulationSlide 2Transcription: 3 DNA-Dependent RNA PolymerasesRelative cellular RNA abundanceRNA Polymerase IISlide 6RNAP II PromotersTATA Box of Class II PromotersUpstream elements: Class II promotersEnhancers and SilencersTranscription factors for Class II promotersSlide 12Eukaryotic Transcription Factors: StructureDNA-binding domainsActivation from a Distance: EnhancersSlide 16Chromatin ModificationSlide 18Slide 19In Vivo StudiesPost-Transcriptional ProcessesCap Functions3’ end Processing & Polyadenylation Mechanism3' End FormationPlant Nuclear Gene Expression & RegulationA lot of steps to regulate:1. Transcription*2. Capping3. 3' maturation, cleavage & polyadenylation4. Splicing*5. Transport to Cytoplasm6. 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 Polymerases1. 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 snRNAs3. 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 II1. 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 proteins3. Does not bind DNA by itself, requires other proteins to bind promoter first!Fig. 6.30, Buchanan et al.TFII – transcription factors for RNA Pol IIRNAPII – RNA Pol IIRNAP 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.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 promotersFound 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 Silencers1. 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 factorsTranscription factors for Class II promoters1. 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.Assembly of the RNA Pol II Initiation Complex= basal factors + RNAP II Fig. 7.45, Buchanan et al.TFIIF delivers Pol IITFIIH PO4ylates the LS of Pol II, allowing it to escape the promoter.Eukaryotic Transcription Factors: Structure•Mostly about factors that bind USEs:1. Modular structure:–DNA-binding domain–Transcription-activating domain2. Can have > 1 of each type of module 3. Many factors also have a dimerization domain (some can form heterodimers).DNA-binding domains1. Zinc – containing modules2. Homeodomains (conserved amino acid seq.)3. bZIP and bHLH motifs4. AP2 (mainly in plants)(not an exhaustive list, just what might be on the test!)Activation from a Distance: Enhancers•3 possible modelsFactor binding induces:1. Supercoiling of the promoter DNA2. Sliding of the complex to the promoter3. Looping out of DNA between enhancer and promoter3 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 core3. Histone H1 on outsideH2BH4H3H1H2BH2ADNAPacking ratio ~5Nucleosome core = octamer of histones (2 each of H2A, H2B, H3, H4) + 2 wraps (145 bp) of DNAFig. 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)In Vivo Studies •Promoters of active genes are often deficient in nucleosomesSV40 virus minichromosomes with a nucleosome-free zone at its twin promoters. Fig. 13.25Can also be shown for cellular genes by DNase I digestion of chromatin – promoter regions are hypersensitive to DNase I.Post-Transcriptional Processes1. Capping2. 3’ end formation (not much regulation of the above steps)3. Splicing – alternative splicing4. Translation – regulate initiation stepCap Functions•Capping also includes methylation of the ribose (2-OH) on nt #1 and sometimes #2. •Cap functions:1. Protection from 5 exoribonucleases2. Enhances translation in the cytoplasm3. Enhances transport from the nucleus4. 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 degraded3' End FormationCIS (elements)•AAUAA is the key signal in higher plants, its found ~20 nt from the polyA-tail.–Other sequences 5' to the AAUAA also