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Gene Expression

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.

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Gene Expression

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  1. Gene Expression

  2. 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.

  3. 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.

  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) Plasmid single copy vs. multicopy plasmids 1. DNA replication 2.Transcription Primary transcript mRNA degradation 3. Posttranscriptional processing Mature mRNA 4. Translation inactive protein Protein degradation 5. Posttranslational processing active protein

  11. Proteins Regulate Gene Expression

  12. Proposed Model rRNA, tRNA etc. Proteins DNA Pre-RNA mRNA

  13. Post-Transcriptional Modification in Eukaryotes • primary transcriptformed 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 • Exons: coding regions • Introns:noncoding regions • Introns are removed by “splicing” GU at 5’ end of intron 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) complementary 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 U1, U2 & U 5 recognize donor and acceptor sites for splicing specificity 2 1 1 2

  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. Condition 2 “turned off” “turned on” 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 25 26 19 20 21 22 23 24 induced gene repressed gene constitutively expressed gene inducible/ repressible genes Gene regulation (1) Condition 1 “turned off” “turned on” Chr. I Chr. II Chr. III

  21. Condition 4 upregulated gene expression down regulated gene expression Gene regulation (2) Condition 3 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 25 26 19 20 21 22 23 24 constitutively expressed gene

  22. Modulators of transcription • Modulators: (1) specificity factors, (2) repressors, (3) activators • Specificity factors: Alter the specificity of RNA polymerase Examples: s-factors (s70, s32 ) s70 s32 Standard promoter Heat shock promoter Housekeeping gene Heat shock gene

  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 Coding sequence Promoter

  24. Negative regulation (1) Repressor Effector Example: lac operon RESULT: Transcription occurs when the gene is derepressed

  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 RNA pol. Activator 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. Pi I P Q1 Z Y A Q3 Q2 The lactose (lac) operon • 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. LacZ LacY LacA Inducer molecules: Allolactose: - natural inducer, degradable IPTG (Isopropylthiogalactoside) - synthetic inducer, not metabolized, lac repressor Pi Pi I I P P Q1 Q1 Z Z Y Y A A Q3 Q3 Q2 Q2 Regulation of the lac operon

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