Fundamentals of Gene Regulation

1. Fundamentals of Gene Regulation The DNA of different tissues and cell types is the same in a specific organism unlike the RNA and protein content. Gene regulation must therefore operate to produce different amounts of different RNA in different cell types, from the same DNA. Rekha Jain HariniChandra

2. PROKARYOTIC EUKARYOTIC Ground state: off Ground state: on Repressed state: off Active state: on Master Layout (Part 1)‏ 1 This animation consists of 3 parts: Part 1 – Basics of gene regulation Part 2 – Gene regulation in bacteria Part 3 – Gene regulation in eukaryotes 2 DNA RNA 3 PROTEIN CENTRAL DOGMA 4 5 Please re-draw figures. Source: Biochemistry by Lehninger, 4th edition (ebook), Genetics by Griffiths, 8th edition (ebook)

3. Definitions of the components:Part 1 – Basics of Gene regulation 1 1. Gene regulation: The process by which the synthesis of a gene’s mRNA transcript and its corresponding protein product is controlled or regulated by various signal molecules is termed as gene regulation. In this process, a cell determines which genes to express and when to express them. Regulation of processes is essential to ensure that no wastage of energy or cellular materials takes place. 2. Single celled organisms: Organisms that have only one cell containing all the organelles and genetic material within one common compartment are known as single celled organisms. The bacterial genome has 4000 genes of which only a fraction of them are expressed at any given time. Moreover, requirements for gene products vary with time such that some products are required in large amounts while others in smaller quantities. 3. Multi-cellular organisms: Gene regulation is a more complex process in eukaryotic and multi-cellular organisms that contain more number of cell organelles, each having complex processes taking place in them. The human genome contains around 35,000 genes, out of which only a fraction of them are expressed in a cell at any given time. Gene expression varies in different cell types even though their copy of the genome is identical. Certain genes, known as housekeeping genes, are expressed in all cells while others are specific only to certain cell types. For example, the gene for glucagon hormone is expressed only in pancreatic cells while antibody synthesis genes are continuously expressed in plasma cells. 4. Transcription: Transcription is the process by which information from a double stranded DNA molecule is converted into a chemically related single stranded RNA molecule by making use of one strand as the template. Transcription which takes place in the eukaryotic nucleus is separated in space and time from translation taking place in the cytoplasm. 5. Translation: A process by which the mRNA sequence is read in the form of three letter codes known as codons to incorporate the corresponding amino acids in the growing polypeptide chain with the active involvement of rRNA, tRNA and several other enzymes. 2 3 4 5

4. Definitions of the components:Part 1 – Basics of Gene regulation 1 6. Different levels of gene regulation: Gene regulation can be carried out at several levels starting from the synthesis of the mRNA transcript till the degradation of its corresponding protein product. The various stages include: • Synthesis of the primary RNA transcript (transcription) • Post-transcriptional modification of mRNA • Messenger RNA (mRNA) degradation • Protein synthesis (translation) • Posttranslational modification of proteins • Protein targeting and transport • Protein degradation 7. Active state: The state in which the gene is turned “on” and synthesizes its corresponding mRNA and protein. 8. Repressed state: The state in which the gene is turned “off” and no transcription occurs. This could be due to binding of a repressor molecule to the gene. 2 3 4 5

5. RNA pol Repressor RNA pol Part 1, Step 1: 1 IN PROKARYOTES Activator protein 2 No transcription Transcription 3 Coding region of gene Ground state: on Repressed state: off 4 Action Description of the action Audio Narration In prokaryotes, transcription by RNA polymerase can take place with the help of an activator protein. However, in the presence of a repressor molecule, the binding site for RNA polymerase is inaccessible due to which transcription does not occur. In the ground state, the repressor does not remain bound because of which the gene is turned “on”. First show the figure on the left with the coloured line & the red oval bound to it. The orange oval must then appear and bind to the red oval & coloured line as depicted. Once this happens, the green arrow & label must appear. Next show the figure on right with coloured region & the yellow shape bound to it. The red oval must now approach the yellow shape and must move away as soon as it reaches the shape followed by appearance of the green arrow with red cross over it. As shown in animation. 5

6. Part 1, Step 2: 1 IN EUKARYOTES Nucleosome – chromatin & histone 2 Enhancer Transcription factors No transcription Promoter proximal element 3 Transcription Ground state: off Active state: on 4 Action Description of the action Audio Narration The regulatory site of DNA in eukaryotes remains inaccessible for binding by transcription machinery due to which gene expression remains turned off in the ground state. In the active state, however, the DNA forms a loop thereby bringing together the promoter proximal element and enhancer . Interactions of these two elements with RNA polymerase successfully activates expression of the gene. PLEASE RE-DRAW ALL FIGURES. First show the two oval structures on top which must then be zoomed into to show the coloured regions below. Once this happens the green arrow with red cross over it must appear. Next show the figure on the right with its labels. This is followed by appearance of the arrow & the label ‘transcription’. As shown in animation. 5 Source: Genetics by Griffiths, 8th edition (ebook) )‏

7. Promoter Operator Gene promoter Gene B Gene C Gene A Master Layout (Part 2)‏ 1 This animation consists of 3 parts: Part 1 – Basics of Gene regulation Part 2 – Gene regulation in prokaryotes Part 3 – Gene regulation in eukaryotes 2 Transcription 3 Positive regulation Transcription Negative regulation Activator mRNA Translation No transcription No transcription Repressor 4 Protein A Protein B Protein C Transcription 5

8. Definitions of the components:Part 2 – Gene regulation in prokaryotes 1 1. Operon: A functioning unit of genomic material that is made up of a cluster of functionally related genes that are under the control of a single regulatory element. Operon arrangements are a commonly observed mechanism of gene regulation in prokaryotes and can be either inducible or repressible. 2. Lacoperon: The first system of gene regulation that was understood in E. coli, worked out by Francois Jacob and Jacques Monod in 1962. The lacoperonis negatively controlled by the lacIrepressor and positively regulated by catabolic activator protein (CAP). 3. Operator: Operatorsare regions of DNA that are around15 nucleotides long and are generally located near a promoter element such that they control the access of RNA polymerase to this region. 4. Promoter: The region of DNA to which RNA Polymerase binds and starts the process of transcription. 5.Activator: A molecule that enhances the interaction between RNA polymerase and a particular promoter region, thereby facilitating expression of the gene. 6. Repressor: A protein that binds to a regulatory region such as an operator,adjacent to a gene and thereby prevents its transcription by impeding the binding of RNA polymerase. 7. Effectors: Effectors are molecules that affect the binding of activators and repressors to the operator region of DNA. These can either be inducers, in case of inducible systems, or a co-repressors if it is a repressible system. 2 3 4 5

9. Definitions of the components:Part 2 – Gene regulation in prokaryotes 1 8. Inducible system: An inducible system is originally ‘off’ in its ground state and must be turned on by an effector molecule, which is known as the inducer. In the negative regulation mechanism, the inducer binds to repressor and prevents it from binding to the operator region. This allows RNA polymerase to proceed with transcription by binding to the promoter. In positive regulation mechanism however, the inducer binds to the inactive activator to produce the active activator molecule which in turn facilitates binding of RNA polymerase to the promoter to turn on expression. 9. Repressible system: The ground state in case of a repressible system is ‘on’ and it has to be turned off by an effector molecule, which is known as the co-repressor. In case of negative regulation mechanism, the co-repressor binds to the inactive repressor molecule and activates it, thereby preventing gene expression. In positive regulation, on the other hand, the co-repressor binds to the activator molecule and prevents its binding to the promoter region, thereby turning off gene expression. 2 3 4 5

10. Repressor Promoter Promoter Operator Operator Gene Gene Part 2, Step 1: 1 Inducible system Inducer Inducer Positive regulation 2 Transcription No transcription Active activator Inactive activator Negative regulation Transcription No transcription 3 4 Action Description of the action Audio Narration An inducible system is ‘off’ in its ground state and must be turned on by an effector molecule, which is known as the inducer. In the negative regulation mechanism, the inducer binds to repressor and prevents it from binding to the operator region. This allows RNA polymerase to proceed with transcription by binding to the promoter. In positive regulation mechanism however, the inducer binds to the inactive activator to produce the active activator molecule which in turn facilitates binding of RNA polymerase to the promoter to turn on expression. As shown in animation. First show the coloured chain with labels on the left followed by binding of ‘inactive activator’ & appearance of the green arrow with red cross saying ‘no transcription’. Next show appearance of the grey square which binds to blue cloud. The cloud must change colour & the text box must show ‘active activator’. The red cross must disappear over the green arrow & must say ‘transcription’. Next, the coloured chain on the right is shown with the yellow hexagon bound to the red region & the arrow with red cross over it. The grey box must bind to this yellow figure & both must be removed along with the red cross. 5

11. Promoter Promoter Operator Operator Gene Gene Part 2, Step 2: 1 Repressible system Co-repressor Co-repressor Positive regulation 2 Activator No transcription Transcription Negative regulation No transcription 3 Transcription Active repressor Inactive repressor 4 Action Description of the action Audio Narration First show the coloured chain with labels on the left followed by binding of ‘activator’ & appearance of the green arrow saying ‘transcription’. Next show appearance of the green star which binds to ‘activator’ and both these shapes must together get dissociated & the red cross must appear over the green arrow saying ‘no transcription’. Next, the coloured chain on the right is shown with the violet star bound to the red region along with the green arrow and ‘transcription’ label. Next the light green star must bind to the violet shape, which must change colour & the text must change to ‘active repressor’. A cross must appear over the green arrow with the label ‘no transcription’. As shown in animation. The ground state in case of a repressible system is ‘on’. It has to be turned off by an effector molecule, which is known as the co-repressor. In case of negative regulation mechanism, the co-repressor binds to the inactive repressor molecule and activates it, thereby preventing gene expression. In positive regulation, the co-repressor binds to the activator molecule and prevents its binding to the promoter region, thereby turning off gene expression. 5

12. lacI Operator lac Z lacY lacA Promoter Part 2, Step 3: 1 The lac operon: negative control by lacI repressor Presence of lactose: de-repression No transcription Absence of lactose: repression Transcription 2 RNA Polymerase p No binding 3 mRNA b-galactosidase Transacetylase Permease Repressor tetramer Inducer binds repressor protein Repressor protein (monomer) Inducer 4 Action Description of the action Audio Narration First show the blue region giving the strand & then the yellow circle. 4 yellow circles must come together & then bind to the green region. The violet oval must attempt to move forward but must be blocked by the yellow circles. The green arrow with red cross must appear. Next the heading must change & the blue square below must bind to the yellow circles which should then attempt to bind to green region but they should not be able to. The violet oval then moves across the entire coloured region till the end & ‘transcription’ heading must appear. The lac operon consists of a group of genes that are responsible for transport and metabolism of lactose sugar in certain bacteria like E. coli. This operon is under negative regulation by the LacI repressor protein. In absence of the inducer, the tetrameric repressor binds to the operator region, thereby preventing transcription by RNA Polymerase. In presence of the inducer, the inducer binds to the repressor protein which then prevents it from binding to the operator and therefore allows gene expression. As shown in animation. 5

13. Part 2, Step 4: lacI Operator lac Z lacY lacA Promoter 1 The lac operon: Positive Control by CAP and catabolite repression Low glucose cAMP ATP Glucose 2 Lactose CAP Bacterial cells cAMP Cell growth & division High glucose Transcription 3 cAMP ATP 4 Action Description of the action Audio Narration First show the blue region giving the strand & then the yellow circle. 4 yellow circles must come together & then bind to the green region. The violet oval must attempt to move forward but must be blocked by the yellow circles. The green arrow with red cross must appear. Next the heading must change & the blue square below must bind to the yellow circles which should then attempt to bind to green region but they should not be able to. The violet oval then moves across the entire coloured region till the end & ‘transcription’ heading must appear. Lacoperon also undergoes positive regulation by means of the cAMP-CAP system. Glucose is the preferred energy source for bacteria and if both glucose and lactose are present, b-galactosidaseenzyme which metabolizes lactose is not synthesized. High glucose levels prevent synthesis of cAMP which is essential for binding to the catabolite activator protein. This protein facilitates transcription of the lacoperon. When glucose levels are low, cAMP is produced which binds to the CAP, which in turn binds to a distal part of the promoter region and facilitates transcription. As shown in animation. 5 Source: Introduction to Genetic Analysis, eight edition (ebook)

14. Master Layout (Part 3)‏ Gene DNA (1) Transcription Primary transcript Nucleotides (2) Post-transcriptional processing (3) mRNA degradation Mature mRNA (4) Translation Protein (inactive)‏ Amino acids (5) Post-translational processing (7) Protein degradation Modified protein (active)‏ (6) Protein targeting and transport 1 This animation consists of 3 parts: Part 1 – Basics of Gene regulation Part 2 – Gene regulation in bacteria Part 3 – Gene regulation in eukaryotes 2 Inaccessible state 3 Chromatin remodeling Accessible state 4 5 Please re-draw all figures. Source: An introduction to Genetic analysis by Griffiths, 8th edition (ebook)‏

15. Definitions of the components:Part 3 – Gene regulation in eukaryotes 1 1. Gene expression: The process of transfer of genetic information from the nucleotide sequence level in a gene to the level of amino acid sequence in a protein or the nucleotide sequence of mRNA is known as gene expression. 2. Eukaryotic regulation: Eukaryotic cells have larger and more complex multimeric regulatory proteins when compared to bacterial cells. The process of regulation is therefore, also more complex and can be achieved either by altering the rate of transcription, the stability of mRNA molecule or through regulation at a translational level. Regulatory elements that control these processes may be tissue specific, thereby activating or deactivating genes only in one kind of tissue. 3. Chromatin: DNA that is packaged with basic proteins known as histonesform a structure known as chromatin in eukaryotes. This chromatin structure helps in restricting access to eukaryotic promoter sites. For gene expression to take place, remodelling of the chromatin must occur wherein, acetylation of histone proteins and demethylation of DNA occur, which then favours transcription. 4. Promoter: The region of DNA to which RNA Polymerase binds and starts the process of transcription. The promoter in eukaryotes contains a sequence of 7bases known as the TATA box, which is bound by a large number of proteins including the TATA-binding protein (TBP), and various transcription factors. 5. Exons: The regions of mRNA that code for specific proteins or entire protein products upon translation are known as exons. They are often discontinuous with intervening nucleic acid sequences being present between them. 6. Introns: The intragenic sequences, sometimes considered as “junk”, that are present in the pre-mRNA but do not get translated into proteins are known as introns. These are removed during the process of RNA splicing. . 2 3 4 5

16. Definitions of the components:Part 3 – Gene regulation in eukaryotes 1 7. Enhancers: Enhancersare sequences of DNA to which regulatory proteins can bind. Most of these are located outside of the promoter region. Binding of transcription factors to enhancers is associated with an increase in the rate of transcription. 8. Silencers: Silencers are control regions of DNA, onto which transcription factors bind in order to decrease the rate of transcription. 9. RNA splicing/post-transcriptional modification: It is the process by which exons are spliced or cut out from the pre-mRNA molecule to give a coding, mature mRNA sequence, which is then translated into protein. This is another point at which gene regulation commonly occurs. Splicing can take place such that the exons are re-joined in different combinations, in a process known as alternative splicing. This allows a variety of different polypeptides to be translated from a single gene. 10. RNA Transport: The process by which only fully processed and mature mRNA is allowed leave the nucleus in order to be translated into protein. Any defective mRNA will be degraded within the nucleus itself. 11. Positive regulation: Although eukaryotic cells exhibit both positive and negative regulatory mechanisms, the positive mechanisms have been found to predominate in all systems characterized so far. The transcriptional ground state is therefore restrictive or silenced, and virtually every eukaryotic gene requires activation before it can be transcribed. 2 3 4 5

17. Gene DNA Part 3, Step 1: 1 LEVELS OF REGULATION OF EUKARYOTIC GENE EXPRESSION (1) Transcription Primary transcript 2 Nucleotides (2) Post-transcriptional processing (3) mRNA degradation Mature mRNA (7) Protein degradation 3 (4) Translation Amino acids (6) Protein targeting and transport (5) Post-translational processing 4 Action Description of the action Audio Narration PLEASE RE_DRAW ALL FIGURES. First show the figure on top appearing followed by the various down arrows and the figures below each arrow. Then show all the backward arrows as depicted in animation above. As shown in animation. Eukaryotic gene regulation is a complex process that can be regulated at various levels starting from the gene transcription to the post-translational modification of the protein to form the active, functional molecule. These levels include transcription, post-transcriptional modification, translation, post-translational modification and protein transport, the most common of which is the transcriptional level. 5 Source: Biochemistry by Lehninger, 4th edition (ebook)‏

18. Part 3, Step 2: 1 Various regulatory proteins G-C rich box 2 mRNA RNA pol II Chromatin remodeling 3 Promoter -proximal element Promoter Regulatory region for transcription in eukaryotes Regulation at transcription level 4 Action Description of the action Audio Narration Chromatin remodelling, which provides accessibility to the gene, is a prerequisite for gene expression and is one of the points of regulation at the transcriptional level. Remodelling involves acetylation of histone proteins and demethylation of DNA which are carried out by various enzymes. Several regulatory proteins are also involved in the transcription process after chromatin remodelling, which serve as important points of regulation. PLEASE RE_DRAW ALL FIGURES. First show the star with text on top followed by the arrow pointing to ‘chromatin remodeling’. The grey figure on top must be shown followed by the arrow showing the figure below. The next heading on right top must appear and the figure on left bottom must then be zoomed into to show the figure on the right. As shown in animation. 5 Source: An introduction to Genetic analysis by Griffiths, 8th edition (ebook)‏

19. Part 3, Step 3: 1 Post-transcriptional regulation 2 Exons 3 A A Addition of Poly(A) tail Poly(A) tail addition Splicing 5’ Capping A A 7-methyl guanosine cap is added to mRNA shortly after the start of transcription, provides recognition by ribosome and protection from RNase A A A A Spliceosome 5’cap Introns 4 Action Description of the action Audio Narration PLEASE RE_DRAW ALL FIGURES. First show the heading on top followed by the three arrows and the headings below that. Then show each figure below these headings in the ovals appearing one after the other as shown. As shown in animation. mRNA synthesized from DNA by transcription undergoes several post-transcriptional modifications to form the mature mRNA which then undergoes translation to form proteins. These modifications are also under regulatory control to moderate the amount of mRNA produced based on the requirement. Addition of a poly (A) tail promotes export of the mRNA from the nucleus and protects the mRNA from degradation. mRNA that does not have these modifications is usually very unstable and will be degraded. 5 Source: An introduction to Genetic analysis by Griffiths, 8th edition (ebook)‏

20. Part 3, Step 4: 1 Masked mRNA is stored in unfertilized sea urchin eggs. These are translated on receiving an appropriate signal and later provide nourishment and to the eggs when required Translational regulation 2 3 Signal Protein A A A A 5’cap Poly(A) tail 4 Action Description of the action Audio Narration PLEASE RE_DRAW ALL FIGURES. First show the heading on top followed by the three arrows and the headings below that. Then show each figure below these headings in the ovals appearing one after the other as shown. As shown in animation. In eukaryotes, the mRNA does not get translated until it receives the appropriate signal, thereby serving as another point of control. Before receiving the signal, enzymes required for the process will not be present and will get synthesized only after the signal is obtained. 5 Source: An introduction to Genetic analysis by Griffiths, 8th edition (ebook)‏; Biochemistry by Lehninger, 4th edition (ebook)‏

21. Part 3, Step 5: 1 Protein degradation Post-translational regulation 2 Protein targeting and transport Post-translation processing 3 Active Protein Inactive Protein 4 Action Description of the action Audio Narration PLEASE RE_DRAW ALL FIGURES. First show the protein structure on the left followed by the arrow and the structure on the right with all the labels as shown. Then show the arrow on the right and elbow arrow on the left with labels. As shown in animation. Proteins undergo several post translational modifications such as phosphorylation, acetylation, methylation, glycosylation etc. These processes are essential for formation of the active, functional protein. Regulation at the post-translational level can lead to degradation of the non-functional protein molecules. 5 Source: Biochemistry by Lehninger, 4th edition (ebook)‏

22. Interactivity option 1:Step No:1 1 Cells growing in their exponential phase in a medium of glucose are transferred to a medium containing only lactose for their carbon source. These cells are again found to have a lag phase before they again start growing rapidly. What could be the possible reason for this? Transfer of cells 2 Glucose Bacterial cells Lactose 3 Medium containing only glucose Medium containing only lactose 4 Results Interacativity Type Options Boundary/limits D is the correct answer,. If user chooses B, it must turn green with the remark ‘correct answer’. If user chooses any of the remaining options, it must turn red with the remark ‘incorrect answer’. User must be allowed to choose any one of the 4 options given in the next slide. Choose the correct option 5

23. Interactivity option 1:Step No:1 1 Cells growing in their exponential phase in a medium of glucose are transferred to a medium containing only lactose for their carbon source. These cells are again found to have a lag phase before they again start growing rapidly. What could be the possible reason for this? A) Due to difference in pH of the two media. 2 B) Due to changes in cell morphology. C) Because of modifications in time required for cell growth and divisions. 3 D) Due to the time it takes to induce the lacoperon which synthesizes b-galactosidase. 4 5

24. Questionnaire 1 1. Negative regulation of the lac operon is brought about by Answers: a) cAMP b) lacIc) CAP d)‏ None of the above 2. Gene regulation is most commonly implemented at which of the following stages? Answers: a) translation b) transcription c) post-translational modification d) protein transport 3. Which of the following is the effector molecule for the repressible system? Answers: a) Activator b) Co-repressor c) Inducer d)‏ Repressor 4. What is the binding interaction that takes place during negative regulation in an inducible system? Answers: a) Inducer-repressorb) Inducer-inactive activator c) Co-repressor – inactive repressor d) Co-repressor- activator 5. The binding site for the effector molecule is? Answers: a) Promoter b) Enhancer c) Operatord)‏ None of the above 2 3 4 5

25. Links for further reading Books: Biochemistry by A.L.Lehninger et al., 4th edition An introduction to Genetics by Griffiths, 8th edition Molecular Biology of the Gene by James Watson, 5th edition