1Developmental Biology - Biology 4361Lecture 8 - Differential Gene ExpressionOctober 13, 2005The principle of genomic equivalence states that all cells in a developing organism have thesame genetic constitution. - therefore, differences in cells (e.g. determination, bias, differentiation) must arise fromdifferential gene expression - all cells express large number of the same genes - “housekeeping” or “common” genes - many cells also express cell-type-specific genes - cell-type-specific gene products include: - bulk products (e.g. ovalbumin, globins); usually from terminally differentiated cells- smaller amounts of regulatory proteins, e.g. transcription factors- crucial in stepwise determination process - cell-type-specific gene expression depends on stage and body position- selective gene transcription depends on regulatory signals- where do regulatory signals come from?- from the egg: maternal stores (e.g mRNAs in the egg) - zygotic de novo expression (i.e. activation of genes expressing regulatoryproteins) of zygotic messagesTranscriptional control - - dependent on regulatory DNA sequences (cis-acting) - and on regulatory proteins (trans-acting)- regulatory proteins can enhance or inhibit transcription- regulatory protein control (sometimes) by intra- and inter-cellular molecular signalsEvidence for transcriptional control - polytene chromosome puffs - variable among different cell types - cDNA libraries from different cells or developmental periods- widely varying suites of messages in different tissues - in situ hybridization- method (see e.g. Kalthoff, method 8.1, p. 179)DNA Sequences Controlling Transcription - gene anatomy (vertebrate): [general convention: DNA - 3'-5' = upstream - downstream; RNA polymerase runs froma 3' to 5' direction on the “sense” strand; produces RNA in a 5' to 3' orientation,matching the “antisense” strand; transcribed genes referred to in 5' - 3' = upstream -2downstream orientation] - promoter region - binding and positioning RNA polymerase - transcription initiation site (contains cap sequence; will receive 5' cap) [Note - byconvention, referred to as “+1” base pair (bp) in gene sequence] - 5' cap = methylated guanosine in opposite polarity to RNA- necessary for ribosome binding- may protect mRNA from exonucleases - translation initiation site (ATG = AUG in RNA)- 5' untranslated region (5' UTR) - space between transcription and translation initiationsites (50 bp in human globin gene); can determine rate at which translation isinitiated - exons (RNA which “exits” nucleus)- coding sequence - introns - intervening sequences- non-coding sequence - translation termination codon (TAA = UAA in RNA); ribosome dissociates - 3' untranslated region (3' UTR); - includes sequence AATAAA - needed for polyadenylation - poly(A) “tail” ~200-300 A - gives stability to mRNA - allows exit from nucleus - permits translation - may protect mRNA from exonucleases - regulatory region - cis-acting sequences upstream of transcription start site- regulatory DNA sequences function to accelerate or inhibit binding of RNA polymerase II to transcription start site- regulation indirect - i.e. - sequences recognized by and bind trans-acting transcription factors- regulate assembly of large protein complex (basic transcription factors)which determines activity of RNA polymerase IIPromoter - binds RNA polymerase[ RNA polymerases- RNA polymerase I - rRNAs- RNA polymerase II - mRNA- RNA polymerase III - tRNA, 5s rRNA, other small nuclear & cytosolic RNAs- others in mitochondria, chloroplasts- RNA polymerase II produces pre-messenger RNA (aka nuclear RNA, nRNA;heterogeneous nuclear RNA)- or just mRNA prior to RNA processing]3 - promoter necessary for all transcription- known as core promoter or basal promoter or proximal promoter- most located directly upstream - 5'- of transcription start site- several consensus sequences (shared sequences) among species- e.g. TATA box; A/T base pairs; usu. located -25 to -30- sequence binds RNA polymerase II- NOTE - no binding without additional transcription factors (eukaryotes)- at least 6 transcription factors necessary- e.g. IIB recognition element - binds transcription factor IIB- others e.g. in TATA-less promoters - contain initiator element, downstreampromoter element - located ~ +30 - basal (general) transcription factors - promoter attracts and binds basal transcription factors (general transcription factors) - general transcription factors bind to core promoters of most genes- ~ 20 general transcription factors- TFIID - recognizes TATA box- subunit (TATA-binding protein = TBP) recognizes TATA sequence- position RNA polymerase molecule near transcription start site - e.g. TFIIA, TFIIB, etc.- TATA box-binding protein (TBP)- TBP-associated factors (TAFs); at least 8 - some genes operate with multiple core promoters- can be regulated by multiple trans-elements- multiple promoters can operate at different levels; e.g.- “weak” or “strong” promoters- slow or rapid RNA polymerase binding = slow or rapid transcription - different combinations of general transcription factors are able to stimulate transcription - tovariable degrees - distribution of general transcription factors fairly equal among cell types - concentration of general transcription factors probably not limiting in any cellEnhancers - increase or inhibit (= silencer) activity of core promoters - distinguished from promoters:1. need promoter to work; promoter works independently2. enhancers effective from a distance away from transcribed gene; promoters always inproximity 3. enhancers are effective in reverse orientation - promoters are not4 - promoters and enhancers are equivalent among all cell in an organism- it follows that selective gene activity must be controlled by differential trans-acting elementsTranscription Factors - functional domains- DNA binding domain- binds to promoter or enhancer- activation domain- interacts with other transcription factors and/or RNA polymerase - many dimeric- may be homodimers or heterodimers- dimerization domain - ligand-binding domain (e.g. estrogen, retinoic acid) - enhancer may bind multiple transcription factors- each will have different effect on assembly or activity of transcription complex- net outcome depends on relative concentration of each competing factor - transcriptional activators and