Chapter 21 (Part 2) Transcriptional Regulation and RNA Processing Gene Expression • Constitutive – Genes expressed in all cells (Housekeeping genes) • Induced – Genes whose expression is regulated by environmental, developmental, or metabolic signals. Regulation of Gene Expression RNA Processing 5’CAP Active enzyme Post-translational modification mRNA AAAAAA RNA Degradation Protein Degradation Transcriptional Regulation • Regulation occurring at the initiation of transcription. • Involves regulatory sequences present within the promoter region of a gene (cis-elements) • Involves soluble protein factors (transacting factors) that promote (activators) or inhibit (repressors) binding of the RNA polymerase to the promoter Cis-elements • Typically found in 5’ untranscribed region of the gene (promoter region). • Can be specific sites for binding of activators or repressors. • Position and orientation of cis element relative to transcriptional start site is usually fixed. Enhancers • Enhancers are a class of cis-elements that can be located either upstream or downstream of the promoter region (often a long distance away). • Enhancers can also be present within the transcribed region of the gene. • Enhancers can be inverted and still function 5’-ATGCATGC-3’ = 5’-CGTACGTA-3’ Activators and Repressors • Activators and repressors- Bind to specific cis-elements • Promote or inhibit assembly of transcriptional initiation complex. • Co-activators and corepressors – bind to proteins associated with cis-elements. co-A A RNAP +1 RNAP co-R R +1 Structural Motifs in DNA-Binding Regulatory Proteins • Crucial feature must be atomic contacts between protein residues and bases and sugar-phosphate backbone of DNA • Most contacts are in the major groove of DNA • 80% of regulatory proteins can be assigned to one of three classes: helix-turn-helix (HTH), zinc finger (Zn-finger) and leucine zipper (bZIP) • In addition to DNA-binding domains, these proteins usually possess other domains that interact with other proteins The Helix-Turn-Helix Motif • contain two alpha helices separated by a loop with a beta turn • The C-terminal helix fits in major groove of DNA; N-terminal helix stabilizes by hydrophobic interactions with Cterminal helix The Zn-Finger Motif Zn fingers form a folded beta strand and an alpha helix that fits into the DNA major groove. The Leucine Zipper Motif • Forms amphipathic alpha helix and a coiled-coil dimer • Leucine zipper proteins dimerize, either as homo- or heterodimers • The basic region is the DNA-recognition site • Basic region is often modeled as a pair of helices that can wrap around the major groove Transcription Regulation in Prokaryotes • Genes for enzymes for pathways are grouped in clusters on the chromosome - called operons • This allows coordinated expression • A regulatory sequence adjacent to such a unit determines whether it is transcribed - this is the ‘operator’ • Regulatory proteins work with operators to control transcription of the genes Induction and Repression • Increased synthesis of genes in response to a metabolite is ‘induction’ • Decreased synthesis in response to a metabolite is ‘repression’ Binding of some trans-factors is regulated by allosteric modification lac operon • Lac operon – encodes 3 proteins involved in galactosides uptake and catabolism. • lacY = Permease – imports galactosides (lactose) • lacZ = b-galactosidase – Cleaves lactose to glucose and galactose. • lacA = thiogalactoside transacetylase – function unclear • Expression of lac operon is negatively regulated by the lacI protein Lactose (Lac) Operon • Diauxic growth • Operon organization • Negative and positive regulation – Repressor (lacI) – CAP (crp) Glucose is E. coli’s primary carbon source. But.. it can grow on different carbon sources. Diauxic growth of E. coli on a mixture of lactose + glucose. • When Glucose runs out but Galactose is present, a set of genes (lac operon) are induced to break down Lactose The lac I protein • The structural genes of the lac operon are controlled by negative regulation • lacI gene product is the lac repressor • When the lacI protein binds to the lac operator it prevents transcription • lac repressor – 2 domains - DNA binding on Nterm; C-term. binds inducer, forms tetramer. Operator and RNA Polymerase Bind at Overlapping Sites Inhibition of repression of lac operon by inducer binding to lacI • Binding of inducer to lacI cause allosteric change that prevents binding to the operator • Inducer is allolactose which is formed when excess lactose is present. Inducer : Allolactose, produced by side reaction of lacZ Lehninger: Principles of Biochemistry, 3rd Ed. IPTG is a Gratuitous Inducer IPTG is a synthetic molecule • Synthetic molecule Not metabolized by lacZ Used by molecular biologist to induce protein expression using the lac promoter Binding of inducer causes allosteric change that prevents binding to operator LacI is always bound to operator unless the inducer is present Although some leaky expression does occur Keeps lac operon in pressed state until it is needed Repression of the Tryptophan operon: A variation of the theme Catabolite Repression of lac Operon (Positive regulation) • When excess glucose is present, the lac operon is repressed even in the presence of lactose. • In the absence of glucose, the lac operon is induced. • Absence of glucose results in the increase synthesis of cAMP • cAMP binds to cAMP regulatory protein (CRP) (AKA CAP). • When activated by cAMP, CRP binds to lac promoter and stimulates transcription. Molecular Cell Biology, 4th Edition, Lodish et. al. (2000) Why does the Lac Operon need an activator? Lac promoter has lousy promoter!!! Post-transcriptional Modification of RNA • tRNA Processing • rRNA Processing • Eukaryotic mRNA Processing tRNA Processing •tRNA is first transcribed by RNA •Polymerase III, is then processed •tRNAs are further processed in the chemical modification of bases rRNA Processing •Multiple rRNAs are originally transcribed as single transcript. •In eukaryotes involves RNA polymerase I •5 endonuclases involved in the processing Processing of Eukaryotic mRNA 5’ Capping • Primary transcripts (aka pre-mRNAs or heterogeneous nuclear RNA) are usually first "capped" by a guanylyl group • The reaction is catalyzed by guanylyl transferase • Capping G residue is methylated at 7position • Additional methylations occur at 2'-O positions of next two residues and at 6amino of the first adenine • Modification required to increase mRNA stability 3'-Polyadenylylation • Termination of transcription occurs only after RNA polymerase has transcribed past a consensus AAUAAA sequence the poly(A)+ addition site • 10-30 nucleotides past this site, a string of 100 to 200 adenine residues are added to the mRNA transcript the poly(A)+ tail • poly(A) polymerase adds these A residues • poly(A) tail may govern stability of the mRNA Splicing of Pre-mRNA • Pre-mRNA must be capped and polyadenylated before splicing • In "splicing", the introns are excised and the exons are sewn together to form mature mRNA • Splicing occurs only in the nucleus • The 5'-end of an intron in higher eukaryotes is always GU and the 3'-end is always AG • All introns have a "branch site" 18 to 40 nucleotides upstream from 3'-splice site Splicing of Pre-mRNA • Lariat structure forms by interaction with 5’splice site G and 2’OH of A in the branch site. • Exons are then joined and lariot is excised. • Splicing complex includes snRNAs that are involved in identification of splice junctions.