1. Gene Expression

2. 2 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.

3. 3 Expression of Genetic Information Production of proteins requires two steps: 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. 4

5. 5 RNA Synthesis DNA template: 3’-to-5’ DNA template: 3’-to-5’ RNA synthesis: 5’-3’; no primer needed RNA synthesis: 5’-3’; no primer needed

6. 6 Protein Coding Genes ORF ORF long (usually >100 aa)long (usually >100 aa) “known” proteins  likely“known” proteins  likely Basal signals Basal signals Transcription, translationTranscription, translation Regulatory signals Regulatory signals Depend on organismDepend on organism  Prokaryotes vs Eukaryotes

7. 7 Gene structure relevant to metabolic regulation

8. 8 Promoters

9. 9 Eukaryotic genes

10. 10 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

11. 11 Regulation of gene expression Plasmid Gene (red) with an intron (green)Promoter 2. Transcription Primary transcript 1. DNA replication 3. Posttranscriptional processing 4. Translation mRNA degradation Mature mRNA 5. Posttranslational processing Protein degradation inactive protein active protein single copy vs. multicopy plasmids

12. 12 Proteins Regulate Gene Expression

13. 13 Proposed Model DNA Pre-RNA mRNA rRNA, tRNA etc. Proteins

14. 14 Post-Transcriptional Modification in Eukaryotes primary transcript formed first primary transcript formed first then processed (3 steps) to form mature mRNA then processed (3 steps) to form mature mRNA then transported to cytoplasm 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

15. 15 Intron Splicing in Eukaryotes Exons: coding regions Exons: coding regions Introns: noncoding regions Introns: noncoding regions Introns are removed by “splicing” Introns are removed by “splicing” GU at 5’ end of intron AG at 3’ end of intron

16. 16 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

17. 17 5’ exon then moves to the 3’ splice acceptor site where a second cut is made by the spliceosome 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 Exon termini are joined and sealed Splicesomes Roles in Spicing out Intron 12 12 1 2 U1, U2 & U 5 recognize donor and acceptor sites for splicing specificity

18. 18 Mode of gene regulations Constitutively expressed genes: 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: Inducible genes: Genes that are transcribed and translated at higher levels in response to an inducing factor Repressible genes: Repressible genes: Genes whose transcription and translation decreases in response to a repressing signal

19. 19 Definitions Housekeeping genes: Housekeeping genes: genes for enzymes of central metabolic pathways (e.g. TCA cycle)genes for enzymes of central metabolic pathways (e.g. TCA cycle) these genes are constitutively expressedthese genes are constitutively expressed the level of gene expression may varythe level of gene expression may vary

20. 20 Genes Can Be Turned On/Off

21. 21 Gene regulation (1) Chr. I Chr. II Chr. III Condition 1 “turned on” “turned off” Condition 2 “turned off” “turned on” 123456789 101112131415161718 192021222324 2526 constitutively expressed gene induced gene repressed gene inducible/ repressible genes

22. 22 Gene regulation (2) constitutively expressed gene 123456789 101112131415161718 192021222324 2526 Condition 3 Condition 4 upregulated gene expression down regulated gene expression

23. 23 Modulators of transcription Modulators: Modulators: (1) specificity factors, (2) repressors, (3) activators 1. Specificity factors: Alter the specificity of RNA polymerase Examples: s-factors (s 70, s 32 )  70  32 Heat shock geneHousekeeping gene Heat shock promoter Standard promoter

24. 24 Modulators of transcription 2. Repressors: mediate negative gene regulation mediate negative gene regulation may impede access of RNA polymerase to the promoter may impede access of RNA polymerase to the promoter actively block transcription actively block transcription bind to specific “operator” sequences (repressor binding sites) bind to specific “operator” sequences (repressor binding sites) Repressor binding is modulated by specific effectors Repressor binding is modulated by specific effectors Coding sequence Repressor Operator Promoter Effector (e.g. endproduct)

25. 25 Negative regulation (1) Repressor Effector Example: lac operon RESULT: Transcription occurs when the gene is derepressed

26. 26 Negative regulation (2) Repressor Effector (= co-repressor) Example: pur-repressor in E. coli; regulates transcription of genes involved in nucleotide metabolism

27. 27 Modulators of transcription 3. Activators: mediate positive gene regulation mediate positive gene regulation bind to specific regulatory DNA sequences (e.g. enhancers) bind to specific regulatory DNA sequences (e.g. enhancers) enhance the RNA polymerase -promoter interaction and actively stimulate transcription enhance the RNA polymerase -promoter interaction and actively stimulate transcription common in eukaryotes common in eukaryotes Coding sequence Activator promoter RNA pol.

28. 28 Positive regulation (1) Activator

29. 29 Positive regulation (2) RNA polymerase ActivatorEffector

30. 30 Operons a promoter plus a set of adjacent genes whose gene products function together. a promoter plus a set of adjacent genes whose gene products function together. usually contain 2 –6 genes, (up to 20 genes) usually contain 2 –6 genes, (up to 20 genes) these genes are transcribed as a polycistronic transcript. these genes are transcribed as a polycistronic transcript. relatively common in prokaryotes relatively common in prokaryotes rare in eukaryotes rare in eukaryotes

31. 31 The lactose (lac) operon Contains several elements Contains several elements lacZ gene = b-galactosidaselacZ gene = b-galactosidase lacY gene = galactosidase permeaselacY gene = galactosidase permease lacA gene = thiogalactoside transacetylaselacA gene = thiogalactoside transacetylase lacI gene = lac repressorlacI gene = lac repressor P i = promoter for the lacI geneP i = promoter for the lacI gene P = promoter for lac-operonP = promoter for lac-operon O 1 = main operatorO 1 = main operator O 2 and O 3 = secondary operator sites (pseudo-operators)O 2 and O 3 = secondary operator sites (pseudo-operators) PiP ZYA I Q3 Q1 Q2

32. 32 Regulation of the lac operon PiP ZYA I Q3 Q1 Q2 Inducer molecules: Allolactose: - natural inducer, degradable IPTG (Isopropylthiogalactoside) - synthetic inducer, not metabolized, lac repressor PiP ZYA I Q3 Q1 Q2 LacZLacYLacA