Prokaryotic gene expression and regulation and Eukaryotic gene expression and regulation

1. DEBRE BIRHAN AGRICULTURAL RESEARCH CENTERDEBRE BIRHAN AGRICULTURAL RESEARCH CENTERDEBRE BIRHAN AGRICULTURAL RESEARCH CENTER Advanced Molecular Biology

2. Coverage 1. Prokaryotic gene expression and regulation Prokaryotic “gene structure” • Basic structure of Operon • Lactose Operon” regulation • Tryptophan Operon” regulation 2. Eukaryotic gene expression and regulation Eukaryotic gene structure Regulons

3. Terminologies Gene expression is the process by which the information on genes is used to synthesize a gene product. Two steps • Transcription • Translation Gene regulation is the process of turning genes on and off to controls the amount and the type of gene products Regulation of gene expression • Controls the developmental process • Responds to environmental stimuli • Helps for adaptation to a novel environmental condition Gene structureis the organization of specialized sequences of genes in the genome.

4. Prokaryotic gene structure

5. Gene regulations

6. A. Basic structure of Operon An operon • Cluster of genes with related functions • Control the gene expression of prokaryotics • Regulate these genes altogether under a single promoter • Transcribed into the same mRNA • Translated simultaneously in ribosome (transcriptional coupling) • Prokaryotics have polycistronicoperon

7. Basic structure of Operon cont’d • Regulatory gene: Encodes a repressor • Promoter The sequence of DNA where RNA polymerase will bind to initiate transcription of the genes that follow • Operator : is the stoplight for RNA polymerase, either allowing or preventing from transcribing all of the structural genes • A series of structural genes : coded gene • A termination sequence: The sequence of DNA which signals the transcription to stop

8. Active and Inactive repressor

9. Operon in prokaryotics There are different types of operon in prokaryotics • Lactose operon: lactose to Glucose & Galactose • Tryptophan Operon: synthesis of tryptophan • Luxoperon; production of luminescent proteins • L-arabinoseoperon- L-arabinose to pentise phosphate pathway , D-xylulose-5-phosphate.

10. I. Lac Operon • The most common example • Catabolic type of operon • E. Coli prefers glucose as source carbon and energy but will metabolize lactose  in the absence of glucose • Contains structural genes that encode enzymes to break down lactose • Activate when Glucose is absent and  lactose is present

11. Lac Operon Regulation • Regulator of lacoperon: • cAMP • CAP • cAMP-CAP complex • Presence or absence of Glucose and Lactose Catabolite repression • Glucose has affinity to bind to enzyme adenylatecyclase • Enzyme adenylatecyclase changes ATP to cAMP • If glucose binds to adenylatecyclase ATP won’t be converted to cAMP

12. Lac Operon Regulation • High percentage of glucose • Preventing the conversion of ATP into cAMP • CAP remain in an inactive conformation • Inactivate lacoperon • Low percentage of glucose • Adenylatecyclase is free and active • cAMP is formed • cAMP-CAP complex formed and activate lacoperon CAP-cAMP complex increases the binding ability of RNA polymerase to the promoter region to initiate the transcription.

13. Catabolite repression Lac Operon got three Enzymes • Lac z- B-galactosidase: cleave lactose into galactose and glucose • Lac y- lactose permease: facilitates the passage of lactose across the phospholipid bi-layer of the cell membrane with an active transport • Lac A- lactose trans acetylase : assist cellular detoxification by acetylating nonmetabolizablepyranosides

14. Glucose repression and cAMP-CAP complex

15. No glucose and no lactose Presence of glucose and no lactose

16. Presence of glucose and lactose The presence of lactose & no glucose

17. II. Tryptophan Operon/trpOperon • The trypoperon in E. coli contains five structural genes corresponding to enzymes that Convert chorismateinto tryptophan • Tryptophan is an amino acid that E. coli  need it to survive for building proteins

18. At high tryptophan concentration

19. At low tryptophan concentration

20. At high concentration of tryptophan • Two tryptophan molecules bind the repressor • The repressor bind to operator sequence • RNA polymerase will be blocked from transcribing the tryptophan genes At low concentration of tryptophan • The repressor protein does not bind to the operator • RNA polymerase can bypass and • The tryptophan genes will be transcribed

21. 2. Eukaryotic Gene Regulation

22. Eukaryotic gene structure • Eukaryotic gene structure is the organization of the eukaryotic genes in the genome. • Immature Transcript contains exonsand intronsregions • At post-transcriptional processing intronsare spliced out by spliceosome • Exon regions are retained in the mature mRNA • Adds a 5' cap to the start of the mRNA • A poly-adenosine tail to the end of the mRNA • These additions stabilise the mRNA and direct its transport from the nucleus to the cytoplasm,

23. 5’ cap • Triphosphatase cut the third phosphate group • Guanyletransferasejoin Guanine to the phosphate remained • Methyl transferasebind methyl group at the seventh N of Guanine • This m7G capping prevents the mRNA from exonuclease attack

24. A poly-adenosine tail • Pre-mrna is first cleaved off by Cleavage and polyadenylation specificity factor (CPSF) • Poly(A) polymerase synthesizes poly(A) tails  • Poly(A)-binding protein II (PAB II) adds Adenine (A) at the 3′ end of mRNA

25. Eukaryotic Gene Structure

26. Eukaryote Gene Regulated • Some of the regulated stages are: • chromatin domains, • transcription, • post-transcriptional modifications, • RNA transport, • translation, and • Post translation/ mRNA degradation.

27. Eukaryotic gene regulation • Eukaryotic genes are regulated in protein-coding sequences and controlling sites called regulon. • unlike operonregulon is a functional genetic unit that composed of a non-contiguous group of genes. • Predominantly regulons are found in eukaryotes. Eg. Adaregulon, CRP regulon, FNR regulon • Eukaryotic gene regulation is more complex • Transcription is conducted in nucleus • Translation occurs in Golgi body or ribosome • Transcription and translation are not coupled

28. Gene expression vs. Gene regulation

29. Regulon vs. Operon • Similarities • Involved in the regulation of gene expression • Composed of DNA • Regulated by inducers, repressors or stimulators.