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Gene Expression Gene expression? Biological processes, such as transcription, and in case of proteins, also translation, that yield a gene product. A gene is expressed when its biological product is present and active. Gene expression is regulated at multiple levels. 2 Expression of Genetic Information Production of proteins requires two steps: Transcription involves an enzyme (RNA polymerase) making an RNA copy of part of one DNA strand. There are four main classes of RNA: i. Messenger RNAs (mRNA), which specify the amino acid sequence of a protein by using codons of the genetic code. ii. Transfer RNAs (tRNA). iii. Ribosomal RNAs (rRNA). iv. Small nuclear RNAs (snRNA), found only in eukaryotes. Translation converts the information in mRNA into the amino acid sequence of a protein using ribosomes, large complexes of rRNAs and proteins. 3 4 RNA Synthesis DNA template: 3’-to-5’ RNA synthesis: 5’-3’; no primer needed 5 Protein Coding Genes ORF • long (usually >100 aa) • “known” proteins likely Basal signals • Transcription, translation Regulatory signals • Depend on organism Prokaryotes vs Eukaryotes 6 Gene structure relevant to metabolic regulation 7 Promoters 8 Eukaryotic genes 9 Eukaryotic gene organization 1. Transcripts begin and end beyond the coding region (5’UTR and 3’UTR) 2. The primary transcript is processed by: 5’ capping 3’ formation / polyA splicing 3. Mature transcripts are transported to the cytoplasm for translation 10 Regulation of gene expression Promoter Gene (red) with an intron (green) single copy vs. multicopy plasmids 1. DNA replication Plasmid 2. Transcription Primary transcript 3. Posttranscriptional processing mRNA degradation Mature mRNA 4. Translation inactive protein 5. Posttranslational processing active protein Protein degradation 11 Proteins Regulate Gene Expression 12 Proposed Model rRNA, tRNA etc. DNA Pre-RNA mRNA Proteins 13 Post-Transcriptional Modification in Eukaryotes primary transcript formed first then processed (3 steps) to form mature mRNA then transported to cytoplasm Step 1: 7- methyl-guanosine “5’-cap” added to 5’ end Step 2: introns spliced out; exons link up Step 3: Poly-A tail added to 3’ end mature mRNA 5’-cap- exons -3’ PolyA tail 14 Intron Splicing in Eukaryotes GU at 5’ end of intron Exons: coding regions Introns: noncoding regions Introns are removed by “splicing” AG at 3’ end of intron 15 Splicesomes Roles in Spicing out Intron RNA splicing occurs in small nuclear ribonucleoprotein particles (snRNPS) in spliceosomes Spliceosomes: protein + small RNAs (U1-8) complementar y to the splice junctions 16 Splicesomes Roles in Spicing out Intron 5’ exon then moves to the 3’ splice acceptor site where a second cut is made by the spliceosome Exon termini are joined and sealed 1 2 2 1 1 2 U1, U2 & U 5 recognize donor and acceptor sites for splicing specificity 17 Mode of gene regulations Constitutively expressed genes: Genes that are actively transcribed (and translated) under all experimental conditions, at essentially all developmental stages, or in virtually all cells. Inducible genes: Genes that are transcribed and translated at higher levels in response to an inducing factor Repressible genes: Genes whose transcription and translation decreases in response to a repressing signal 18 Definitions Housekeeping genes: • genes for enzymes of central metabolic pathways (e.g. TCA cycle) • these genes are constitutively expressed • the level of gene expression may vary 19 Genes Can Be Turned On/Off 20 Gene regulation (1) Condition 2 1 Chr. I 1 10 Chr. II Chr. III 2 19 “turned “turned “turned off” off” on” on” 4 5 6 7 8 3 11 12 20 21 22 constitutively expressed gene 13 14 15 16 23 induced gene 24 9 17 25 18 26 repressed gene inducible/ repressible genes 21 Gene regulation (2) Condition 43 upregulated gene expression 1 2 10 19 down regulated gene expression 3 4 11 12 20 21 22 5 7 8 13 14 15 16 17 23 6 24 25 9 18 26 constitutively expressed gene 22 Modulators of transcription Modulators: (1) specificity factors, (2) repressors, (3) activators 1. Specificity factors: Alter the specificity of RNA polymerase Examples: s-factors (s70, s32 ) s70 Standard Housekeeping gene promoter s32 Heat shock Heat shock gene promoter 23 Modulators of transcription 2. Repressors: mediate negative gene regulation may impede access of RNA polymerase to the promoter actively block transcription bind to specific “operator” sequences (repressor binding sites) Repressor binding is modulated by specific effectors Effector (e.g. endproduct) Repressor Operator Promoter Coding sequence 24 Negative regulation (1) Repressor RESULT: Transcription occurs when the gene is derepressed Effector Example: lac operon 25 Negative regulation (2) Repressor Effector (= co-repressor) Example: pur-repressor in E. coli; regulates transcription of genes involved in nucleotide metabolism 26 Modulators of transcription 3.Activators: mediate positive gene regulation bind to specific regulatory DNA sequences (e.g. enhancers) enhance the RNA polymerase -promoter interaction and actively stimulate transcription common in eukaryotes Activator RNA pol. promoter Coding sequence 27 Positive regulation (1) Activator 28 Positive regulation (2) Activator Effector RNA polymerase 29 Operons a promoter plus a set of adjacent genes whose gene products function together. usually contain 2 –6 genes, (up to 20 genes) these genes are transcribed as a polycistronic transcript. relatively common in prokaryotes rare in eukaryotes 30 The lactose (lac) operon Pi I Q3 P Q1 Z Q2 Y A Contains several elements • • • • lacZ gene = b-galactosidase lacY gene = galactosidase permease lacA gene = thiogalactoside transacetylase lacI gene = lac repressor • • • • Pi = promoter for the lacI gene P = promoter for lac-operon O1 = main operator O2 and O3 = secondary operator sites (pseudo-operators) 31 Regulation of the lac operon Pi Pi II Q3 Q3 PP Q1 Q1 ZZ Q2 Q2 LacZ YY LacY AA LacA Inducer molecules: lac repressor Allolactose: - natural inducer, degradable IPTG (Isopropylthiogalactoside) - synthetic inducer, not metabolized, 32