1. Control of Gene Expression

2. Steps of gene expression Transcription – DNA is read to make a mRNA in the nucleus of our cells 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 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. Prokaryotic Gene Expression PromoterCistron1Cistron2CistronNTerminator TranscriptionRNA Polymerase mRNA 5’3’ Translation Ribosome, tRNAs, Protein Factors 12N Polypeptides N C N C N C 123 Expression mainly by controlling transcription

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. 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 Regulatory elements of transcription

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. The lactose (lac) operon Contains several elementsContains several elements –lacZ gene = β-galactosidase –lacY gene = galactosidase permease –lacA gene = thiogalactoside transacetylase –lacI gene = lac repressor –P i = promoter for the lacI gene –P = promoter for lac-operon –Q 1 = main operator –Q 2 and Q 3 = secondary operator sites (pseudo-operators ) PiP ZYA I Q3 Q1 Q2

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

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 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. The regulation or feedback on gene expression is how the cell’s metabolism is controlled. This regulation can happen in different ways: 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. – –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 Other proteins DNA Transcription factors Activator proteins RNA polymerase Promoter Gene 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. Gene regulation of the transcription 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

25. Gene regulation constitutively expressed gene 123456789 101112131415161718 192021222324 2526 Condition 3 Condition 4 upregulated gene expression down regulated gene expression

26. Post-Transcriptional Modification in Eukaryotes 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

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

28. Cap Functions 1. 1.Protection of the mRNA from degradation (Protection from 5 exoribonucleases) 2. 2.Enhances translation in the cytoplasm (Enhancement of the mRNA’s translatability) 3. 3.Enhances transport from the nucleus 4. 4.Proper splicing of the pre-mRNA (Enhances splicing of the first intron (for some pre-mRNAs)) The attachment of 7 Me -GTP to the 5’ end of a nascent mRNA with a 5’ to 5’ phospho-ester linkage

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

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

32. Polyribosome incoming large subunit incoming small subunit polypeptide mRNA 1234567 Groups of ribosomes reading same mRNA simultaneously producing many proteins (polypeptides).

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

34. Coupled transcription and translation in bacteria

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

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