Understanding Gene Expression Control Mechanisms in Prokaryotes and Eukaryotes

1. Control of Gene Expression

2. Steps of gene expression • Transcription – DNA is read to make a mRNA in the nucleus of our cells • Translation – Reading the mRNA to make a protein in the cytoplasm Mainly controlled at the level of transcription

3. Prokaryotic and eukaryotic gene organization Prokaryotic transcriptional regulatory regions (promoters and operators) lie close to the transcription start site Functionally related genes are frequently located near each other These “operons” are transcribed into a single mRNA with internal translation initiation sites

4. Ribosome, tRNAs, Protein Factors Translation Prokaryotic Gene Expression Expression mainly by controlling transcription Promoter Cistron1 Cistron2 CistronN Terminator Transcription RNA Polymerase mRNA 5’ 3’ 1 2 N N N C N C C 1 2 3 Polypeptides

5. Operons • A cluster of related genes often coding for enzymes in a metabolic pathway, which are under the control of a single promoter regulatory region • Genes that work together are located together • A promoter plus a set of adjacent genes whose gene products function together. • They are controlled as a unit • They usually contain 2 –6 genes (up to 20 genes) • These genes are transcribed as a polycistronic transcript. • It is relatively common in prokaryotes • It is rare in eukaryotes

6. Operon System

7. Regulatory elements of transcription Structural genes : DNA that code for a specific polypeptide (protein) Promoter : DNA segment that recognizes RNA polymerase Operator : Element that serves as a binding site for an inhibitor protein (modulator) that controls transcription Repressor : Protein which binds to a specific DNA sequences to determine the transcription of a particular gene Regulatory gene : Gene encode for repressor protein

8. Regulatory gene:Organization of operon

9. Operons • The Tryptophan Operon (Repressible and attenuation) Repressor does not bind to operator unless it interacts with co repressor Biosynthetic pathways • The Lactose Operon (Induction and catabolite repression) Repressor is bound to operator unless molecule to be metabolized is present (inducer) Catabolic pathways

10. A repressible operon

11. Inducible Operon

12. Lactose Operon It codes for the enzymes responsible for lactose catabolism Within the operon, there are three genes that code for proteins (structural protein) and an upstream control region including promoter and a regulatory site called the operator Laying outside the operon is the repressor gene, which codes for a protein (lac repressor) that binds to the operator site and is responsible for the suppression of the operon by blocking the binding of RNA polymerase Transcribed mRNA may contain information for more than one protein (a polycistronic mRNA) The synthesis of these mRNA is regulated in accordance with the needs of the cells at any time thus enable the cell to adapt quickly to changing environmental conditions

13. Pi I P Q1 Z Y A Q3 Q2 The lactose (lac) operon • Contains several elements • lacZ gene = β-galactosidase • lacY gene = galactosidasepermease • lacA gene = thiogalactosidetransacetylase • lacI gene = lacrepressor • Pi = promoter for the lacI gene • P = promoter for lac-operon • Q1 = main operator • Q2 and Q3 = secondary operator sites (pseudo-operators)

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

15. The lac operon: model for gene expression • Includes three protein synthesis coding region--sometimes called "genes" as well as region of chromosome that controls transcription of genes • Genes for proteins involved in the catabolism or breakdown of lactose • When lactose is absent, no transcription of gene since no need for these proteins • When lactose is present, transcription of genes takes place so proteins are available to catalyze breakdown of lactose

16. Eukaryotic gene

17. Eukaryotic gene Expression 1.Transcripts begin and end beyond the coding region 2.The primary transcript is processed by: 5’ capping 3’ formation / polyA splicing 3.Mature transcripts are transported to the cytoplasm for translation

18. Control of Gene Expression

19. Regulation of gene expression • Gene expression is regulated—not all genes are constantly active and having their protein produced • The regulation or feedback on gene expression is how the cell’s metabolism is controlled. • This regulation can happen in different ways: • 1. Transcriptional control (in nucleus): • e.g. chromatin density and transcription factors • 2. Posttranscriptional control (nucleus) • e.g. mRNA processing • 3. Translational control (cytoplasm) • e.g. Differential ability of mRNA to bind ribosomes • 4. Posttranslational control (cytoplasm) • e.g. changes to the protein to make it functional

20. 0 • Regulatory proteins that bind to control sequences • Transcription factors promote RNA polymerase binding to the promoter • Activator proteins bind to DNA enhancers and interact with other transcription factors • Silencers are repressors that inhibit transcription • Control sequences • Promoter • Enhancer • Related genes located on different chromosomes can be controlled by similar enhancer sequences

21. Enhancers 0 Promoter Gene DNA Activator proteins Transcription factors Other proteins RNA polymerase Bending of DNA Transcription

22. Transcription control • Transcription factors • Proximal activators • Distal control elements (enhancers) • DNA binding domain • Activation domains bind to other proteins • These are cell-specific • A few common structures, but found in different combinations in different cells

23. Eukaryotic gene expression

24. 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 of the transcription Condition 1 “turned off” “turned on” Chr. I Chr. II Chr. III

25. Condition 4 upregulated gene expression down regulated gene expression Gene regulation 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

26. 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

27. Alternative picture: co-transcriptional pre-mRNA processing

28. Cap Functions The attachment of 7Me-GTP to the 5’ end of a nascent mRNA with a 5’ to 5’ phospho-ester linkage • Protection of the mRNA from degradation (Protection from 5 exoribonucleases) • Enhances translation in the cytoplasm (Enhancement of the mRNA’s translatability) • Enhances transport from the nucleus • Proper splicing of the pre-mRNA (Enhances splicing of the first intron (for some pre-mRNAs))

29. Intron Splicing Step by step removal of pre-mRNA and joining of remaining exons Removing intron from pre-mRNA • Exons: coding regions • Introns :noncoding regions

30. Polyadenylation The process of adding poly(A) to RNA Synthesis of the poly (A) tail involves cleavage of its 3’end and then the addition of about 40-200 adenine residues to form a poly (A) tail Function - Poly(A) enhances both the lifetime and translatability of mRNA

31. aa5 aa4 aa3 aa2 aa199 aa1 aa200 End Product • The end products of protein synthesis is a primary structure of a protein. • A sequence of amino acid bonded together by peptide bonds.

32. incoming large subunit 1 2 3 4 5 6 7 mRNA incoming small subunit polypeptide Polyribosome Groups of ribosomes reading same mRNA simultaneously producing many proteins (polypeptides).

33. TYPES OF PROTEINS • Enzymes (Helicase) • Carrier (Haemoglobine) • Immunoglobulin (Antibodies) • Hormones (Steroids) • Structural (Muscle) • Ionic (K+,Na+)

34. Coupled transcription and translation in bacteria

35. VALINE original base triplet in a DNA strand a base substitution within the triplet (red) As DNA is replicated, proofreading enzymes detect the mistake and make a substitution for it: POSSIBLE OUTCOMES: OR One DNA molecule carries the original, unmutated sequence The other DNA molecule carries a gene mutation VALINE PROLINE THREONINE LEUCINE GLUTAMATE HISTIDINE

36. A summary of transcription and translation in a eukaryotic cell