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TOPIC : REPLICATION, TRANSCRIPTION , TRANSLATION, GENETIC CODE There are some website references for the processes mentioned above. ASSIGNMENT IS TO BE SUBMITTED FOR EVALUATION Explain pointwise the steps occurring in prokaryotes for - REPLICATION = 20 points and 2 figures, - TRANSCRIPTION = 15 points and 2 figures - TRANSLATION in prokaryotes = 25 points and 4 figures Explain the relevance of GENETIC CODE = 10 points and 1 figure 際際滷 share for replication, transcription and translation /abhijedi123/replication-transcription-translation-and-its-regulation 42 slides, EASY and BRIEF /abhijedi123/replication-transcription-translation-and-its-regulation 55 slides, starts from basic structure , all three processes in brief , https://ocw.mit.edu/courses/biological-engineering/20-106j-systems-microbiology-fall- 2006/lecture-notes/slides07.pdf 90 slides, only replication, transcription and translation EXPLAINED in detail Oral presentation : assignment on replication, transcription and translation Groups Group I : REPLICATION 1 Nuzhat : Semiconservative mode 3 Vatsalya : Dna A + Helicase (Prokaryotes), DNA polymerase (Eukaryores) 8 Chetna : Replication fork, SSB, Gyrase, Topoisomerase, direction, reaction 9 Gaurav : Primase functioning, leading and lagging strands, Okazaki fragments, joining 15 Kishan : termination steps, Tus protein, telomerase 16 Harshit : Types of Polymerases I,II,III,IV,V and 留,硫,粒,隆,竜 Group II : Transcription
4 Simranpreet Kaur : Direction, requiremments 5 Neha : RNA polymerase factor, holoenzyme 10 Swapnil : Promoter site, TATA box, Initiation, binding factors 11 Bhumika : elongation 12 Jyoti : Termination extrinsic and intrinsic types 17 Shikhar : capping and poly A tail formation 21 Aakriti : Prokaryotic regulation, P,O, and other +ve control elements Group III Genetic code and translation 6 Aditya : Genetic code 7 Karan : Ribosome , Prokaryotis and eukaryotic 13 Manish : Initiation factor in prokaryotes, IF1, IF2,IF3 14 Archana :A site, P site, E site, Making complex with three types of RNAs 18 Jigyasa : Initiation for f-met tRNA and new amino acid on A and P site 19 Anamika : peptide bond formation by ribozyme 20 Savitri : Elongation factors,and role of GTP 22 Simran Chaudhri : termination factors, codons and process Regulation of prokaryotic gene expression 1. Definition of gene : it is the basic physical unit of heredity ; linear sequence of nucleotides along a segment of DNA; that provides the coded instructions for synthesis of RNA; when translated into protein this RNA translation leads to the expression of hereditary character 2. The prokaryotic DNA is circular supercoiled chromosome. Proteins are encoded together in blocks called operons. Like : genes needed to use lactose are coded in the lactose (or lac) operon. 3. Gene expression is the combined process of the transcription of : (i) transcription of a gene into mRNA (ii) processing of that mRNA and (iii) its translation into protein 4. Gene activity is controlled first and foremost at the level of transcription through proteins that have specific DNA binding sites. This can have a positive or negative effect on transcription. 5. Control of gene expression occurs at many levels like (i) gene amplification, gene rearrangement , (ii) tissue- specific transcription (iii) posttranscriptional modifications and
(iv) RNA stabilization. 6. There are three types of genes regulation (i) Positive : When gene expression is quantitatively increased by presence of a specific regulatory element called as regulator or activator (ii) Negative : When gene expression is diminished by presence of a specific regulatory element called as repressor or negative regulator (iii) Double negative : An effector that inhibits the function of a negative regulator will bring about a positive regulation . (iv) Repressors and activators are proteins produced in the cell. 7. Types of genes responsible for Gene Expression (i) Inducible gene- whose expression increases in response to an inducer or activator. Inducible genes have relatively low basal rates of transcription. (ii) Constitutive genes ( housekeeping genes) -are expressed at a reasonably constant rate and are not known to be subjected to regulation 8. Significance of Regulation of Gene Expression (i) It is one way to regulate metabolism. It is necessary for Prokaryotes who must use substances and synthesize macromolecules just fast enough to meet their needs (ii) The genes for metabolizing enzymes are expressed only in presence of nutrients. If the enzymes are not needed, their genes are turned off to conserve cells resources. 9. Some important terms related to gene expression (i) cistron : the smallest unit of gene expression. It is the genetic unit coding for a subunit of a protein molecule. If a protein has two or more non identical subunits, one cistron would be for one subunit. (ii) polycistronic mRNA : A single mRNA carrying information for multiple proteins ( e.g. In case of lac operon - the polycistronic mRNA is translated into three separate proteins (iii) Operon : collection of genes involved in a pathway that are transcribed together as a single mRNA in prokaryotic cells. (iv) lac operon : operon in prokaryotic cells that encodes genes required for processing and intake of lactose, when glucose is not available (v) trp operon : series of genes necessary to synthesize tryptophan in prokaryotic cells (vi) inducible operon : operon that can be activated or repressed depending on cellular needs and the surrounding environment (vii) promoter : single regulatory region (nucleotide sequence on operon) that regulate the Operon (viii) operator : region of DNA outside the promoter region that binds activators or repressors (ix) activator : protein that binds to prokaryotic operators to increase transcription. In general, activators bind to the promoter site
(x) repressor : protein that binds to the operator of prokaryotic genes to prevent transcription. In general, repressors bind to operator regions. (xi) positive regulator : protein that increases transcription (xii) negative regulator : protein that prevents transcription (xiii) transcriptional start site : site at which transcription begins (xiv) catabolite activator protein (CAP) : protein that complexes with cAMP to bind to the promoter sequences of operons 10. Lac Operon Model : shows both DOUBLE NEGATIVE control & POSITIVE control 1) Early insights into mechanisms of transcriptional regulation came from studies of E. coli by researchers Francois Jacob & Jacques Monod. 2) Basic concept : Bacteria such as E. coli usually rely on glucose as their source of carbon and energy . When glucose is scarce, E. coli can use lactose as their carbon source even though lactose use is not a major metabolic pathway. 3) An essential enzyme in the metabolism of lactose is 硫-galactosidase, which hydrolyzes lactose into galactose and glucose 4) When glucose or glycerol are available as Carbon source, the growing E. coli cell contains < 10 molecules of 硫 galactosidase molecules. BUT if lactose is provided as carbon source for growth of E. coli cells , quantity of 硫 galactosidase enzyme molecules increase to > several thousands. The synthesis of new enzyme molecules occurs (not the activation of preexisting precursor). 5) Lac Operon is inducible so it gets induced for new synthesis of 硫 galactosidase enzyme 6) Along with the new synthsis of 硫 galactosidase enzyme , the new synthesis of two more enzymes permease, and transacetylase also occurs : Enzyme permease : transports lactose into the cell across the cell membrane Enzyme Galactoside acetyltransferase or transacetylase : it transfers an acetyl group from acetyl-CoA to galactosides, glucosides and lactosides. Acetyl transferase protects cells from building of toxic product created by Beta-galactosidase acting
other galactosides. By acetylating galactosides other than lactose, the transferase prevent Beta-galactosidase 7) Components of lac (lactose) Operon : There are i, z, y, a genes for transcription and o, p sites for binding of two different proteins - the regulator gene i has its gene product as repressor protein. It is Constitutive gene to synthesize lac repressor constantly. - another regulatory DNA sequence ( not a gene because it is not transcribed and has no gene product) o is the operator site, for binding of the repressor. Binding of repressor to the operator prevents transcription of structural genes - p is a promoter site (between i and o) which directs RNA polymerase to the correct transcription initiation site 8) The z, y, a genes of lac operon are structural genes for 硫 - galactosidase, permease, and transacetylase, respectively. These are transcribed to give a single mRNA molecule which after translation would synthesize all three enzyme proteins. Hence the transcription product of the three structural genes i.e. mRNA would be considered as polycistronic mRNA 9) Functioning of lac operon This figure here shows expression of three structural genes and synthesis of their gene products - the three enzymes - as a result of no attachment of repressor protein at the o site when lactose in the medium binds to it.
(i) In absence of lactose, the repressor- in tetrameric form- binds very tightly and rapidly to the operator. Binding of repressor to the operator prevents transcription of structural genes. So the lac operon remains REPRESSED (ii) The presence of lactose triggers functioning of lac operon. Lactose or lactose analogue act as inducer (to switch on) of lac Operon. It means that the INDUCER DEREPRESSES THE LAC OPERON and allows transcription of structural genes for 硫- galactosidase, galactoside permease, and thiogalactoside transacetylase (iii) When inducer (Lactose or lactose analogue) binds to lac repressor, the lac repressor (in a inducer bound condition) is not capable of binding to the operator site. (iv) With the operator site unoccupied, RNA polymerase can bind to the promoter site. The transcription of structural genes begins. Thus E.coli can efficiently utilize lactose for its growth (v) DEREPRESSION OF lac OPERON is a Double negative control. (vi) Figure below explains (a) repression and (b) derepression conditions of the lac operon. There is repression when the movement/ activity of RNA polymerase is not possible as shown in (a).
(vii) Positive control of lac operon : is possible when transcription of the lac operon is stimulated due to CAP/CRP. (viii) lac operon requires the presence of a specific activator protein called catabolite activator protein (CAP), also called cAMP receptor protein (CRP). It is a dimeric protein that first complexes with 35-cyclic AMP (cAMP) and then binds to the lac promoter just upstream from the binding site for RNA polymerase. (ix) This step increases the binding of RNA polymerase and so stimulates the initiation of transcription by approximately a factor of 50. An increase in the cAMP level inside an E. coli bacterium results in the formation of CAP-cAMP complexes. So CAP-cAMP acts as a positive regulator.
Watch an animated tutorial about the workings of lac operon here. https://www.openstax.org/l/lac_operon end

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presentations_for_replication_transcription_translation_and_lac_operon_lecture.docx

  • 1. TOPIC : REPLICATION, TRANSCRIPTION , TRANSLATION, GENETIC CODE There are some website references for the processes mentioned above. ASSIGNMENT IS TO BE SUBMITTED FOR EVALUATION Explain pointwise the steps occurring in prokaryotes for - REPLICATION = 20 points and 2 figures, - TRANSCRIPTION = 15 points and 2 figures - TRANSLATION in prokaryotes = 25 points and 4 figures Explain the relevance of GENETIC CODE = 10 points and 1 figure 際際滷 share for replication, transcription and translation /abhijedi123/replication-transcription-translation-and-its-regulation 42 slides, EASY and BRIEF /abhijedi123/replication-transcription-translation-and-its-regulation 55 slides, starts from basic structure , all three processes in brief , https://ocw.mit.edu/courses/biological-engineering/20-106j-systems-microbiology-fall- 2006/lecture-notes/slides07.pdf 90 slides, only replication, transcription and translation EXPLAINED in detail Oral presentation : assignment on replication, transcription and translation Groups Group I : REPLICATION 1 Nuzhat : Semiconservative mode 3 Vatsalya : Dna A + Helicase (Prokaryotes), DNA polymerase (Eukaryores) 8 Chetna : Replication fork, SSB, Gyrase, Topoisomerase, direction, reaction 9 Gaurav : Primase functioning, leading and lagging strands, Okazaki fragments, joining 15 Kishan : termination steps, Tus protein, telomerase 16 Harshit : Types of Polymerases I,II,III,IV,V and 留,硫,粒,隆,竜 Group II : Transcription
  • 2. 4 Simranpreet Kaur : Direction, requiremments 5 Neha : RNA polymerase factor, holoenzyme 10 Swapnil : Promoter site, TATA box, Initiation, binding factors 11 Bhumika : elongation 12 Jyoti : Termination extrinsic and intrinsic types 17 Shikhar : capping and poly A tail formation 21 Aakriti : Prokaryotic regulation, P,O, and other +ve control elements Group III Genetic code and translation 6 Aditya : Genetic code 7 Karan : Ribosome , Prokaryotis and eukaryotic 13 Manish : Initiation factor in prokaryotes, IF1, IF2,IF3 14 Archana :A site, P site, E site, Making complex with three types of RNAs 18 Jigyasa : Initiation for f-met tRNA and new amino acid on A and P site 19 Anamika : peptide bond formation by ribozyme 20 Savitri : Elongation factors,and role of GTP 22 Simran Chaudhri : termination factors, codons and process Regulation of prokaryotic gene expression 1. Definition of gene : it is the basic physical unit of heredity ; linear sequence of nucleotides along a segment of DNA; that provides the coded instructions for synthesis of RNA; when translated into protein this RNA translation leads to the expression of hereditary character 2. The prokaryotic DNA is circular supercoiled chromosome. Proteins are encoded together in blocks called operons. Like : genes needed to use lactose are coded in the lactose (or lac) operon. 3. Gene expression is the combined process of the transcription of : (i) transcription of a gene into mRNA (ii) processing of that mRNA and (iii) its translation into protein 4. Gene activity is controlled first and foremost at the level of transcription through proteins that have specific DNA binding sites. This can have a positive or negative effect on transcription. 5. Control of gene expression occurs at many levels like (i) gene amplification, gene rearrangement , (ii) tissue- specific transcription (iii) posttranscriptional modifications and
  • 3. (iv) RNA stabilization. 6. There are three types of genes regulation (i) Positive : When gene expression is quantitatively increased by presence of a specific regulatory element called as regulator or activator (ii) Negative : When gene expression is diminished by presence of a specific regulatory element called as repressor or negative regulator (iii) Double negative : An effector that inhibits the function of a negative regulator will bring about a positive regulation . (iv) Repressors and activators are proteins produced in the cell. 7. Types of genes responsible for Gene Expression (i) Inducible gene- whose expression increases in response to an inducer or activator. Inducible genes have relatively low basal rates of transcription. (ii) Constitutive genes ( housekeeping genes) -are expressed at a reasonably constant rate and are not known to be subjected to regulation 8. Significance of Regulation of Gene Expression (i) It is one way to regulate metabolism. It is necessary for Prokaryotes who must use substances and synthesize macromolecules just fast enough to meet their needs (ii) The genes for metabolizing enzymes are expressed only in presence of nutrients. If the enzymes are not needed, their genes are turned off to conserve cells resources. 9. Some important terms related to gene expression (i) cistron : the smallest unit of gene expression. It is the genetic unit coding for a subunit of a protein molecule. If a protein has two or more non identical subunits, one cistron would be for one subunit. (ii) polycistronic mRNA : A single mRNA carrying information for multiple proteins ( e.g. In case of lac operon - the polycistronic mRNA is translated into three separate proteins (iii) Operon : collection of genes involved in a pathway that are transcribed together as a single mRNA in prokaryotic cells. (iv) lac operon : operon in prokaryotic cells that encodes genes required for processing and intake of lactose, when glucose is not available (v) trp operon : series of genes necessary to synthesize tryptophan in prokaryotic cells (vi) inducible operon : operon that can be activated or repressed depending on cellular needs and the surrounding environment (vii) promoter : single regulatory region (nucleotide sequence on operon) that regulate the Operon (viii) operator : region of DNA outside the promoter region that binds activators or repressors (ix) activator : protein that binds to prokaryotic operators to increase transcription. In general, activators bind to the promoter site
  • 4. (x) repressor : protein that binds to the operator of prokaryotic genes to prevent transcription. In general, repressors bind to operator regions. (xi) positive regulator : protein that increases transcription (xii) negative regulator : protein that prevents transcription (xiii) transcriptional start site : site at which transcription begins (xiv) catabolite activator protein (CAP) : protein that complexes with cAMP to bind to the promoter sequences of operons 10. Lac Operon Model : shows both DOUBLE NEGATIVE control & POSITIVE control 1) Early insights into mechanisms of transcriptional regulation came from studies of E. coli by researchers Francois Jacob & Jacques Monod. 2) Basic concept : Bacteria such as E. coli usually rely on glucose as their source of carbon and energy . When glucose is scarce, E. coli can use lactose as their carbon source even though lactose use is not a major metabolic pathway. 3) An essential enzyme in the metabolism of lactose is 硫-galactosidase, which hydrolyzes lactose into galactose and glucose 4) When glucose or glycerol are available as Carbon source, the growing E. coli cell contains < 10 molecules of 硫 galactosidase molecules. BUT if lactose is provided as carbon source for growth of E. coli cells , quantity of 硫 galactosidase enzyme molecules increase to > several thousands. The synthesis of new enzyme molecules occurs (not the activation of preexisting precursor). 5) Lac Operon is inducible so it gets induced for new synthesis of 硫 galactosidase enzyme 6) Along with the new synthsis of 硫 galactosidase enzyme , the new synthesis of two more enzymes permease, and transacetylase also occurs : Enzyme permease : transports lactose into the cell across the cell membrane Enzyme Galactoside acetyltransferase or transacetylase : it transfers an acetyl group from acetyl-CoA to galactosides, glucosides and lactosides. Acetyl transferase protects cells from building of toxic product created by Beta-galactosidase acting
  • 5. other galactosides. By acetylating galactosides other than lactose, the transferase prevent Beta-galactosidase 7) Components of lac (lactose) Operon : There are i, z, y, a genes for transcription and o, p sites for binding of two different proteins - the regulator gene i has its gene product as repressor protein. It is Constitutive gene to synthesize lac repressor constantly. - another regulatory DNA sequence ( not a gene because it is not transcribed and has no gene product) o is the operator site, for binding of the repressor. Binding of repressor to the operator prevents transcription of structural genes - p is a promoter site (between i and o) which directs RNA polymerase to the correct transcription initiation site 8) The z, y, a genes of lac operon are structural genes for 硫 - galactosidase, permease, and transacetylase, respectively. These are transcribed to give a single mRNA molecule which after translation would synthesize all three enzyme proteins. Hence the transcription product of the three structural genes i.e. mRNA would be considered as polycistronic mRNA 9) Functioning of lac operon This figure here shows expression of three structural genes and synthesis of their gene products - the three enzymes - as a result of no attachment of repressor protein at the o site when lactose in the medium binds to it.
  • 6. (i) In absence of lactose, the repressor- in tetrameric form- binds very tightly and rapidly to the operator. Binding of repressor to the operator prevents transcription of structural genes. So the lac operon remains REPRESSED (ii) The presence of lactose triggers functioning of lac operon. Lactose or lactose analogue act as inducer (to switch on) of lac Operon. It means that the INDUCER DEREPRESSES THE LAC OPERON and allows transcription of structural genes for 硫- galactosidase, galactoside permease, and thiogalactoside transacetylase (iii) When inducer (Lactose or lactose analogue) binds to lac repressor, the lac repressor (in a inducer bound condition) is not capable of binding to the operator site. (iv) With the operator site unoccupied, RNA polymerase can bind to the promoter site. The transcription of structural genes begins. Thus E.coli can efficiently utilize lactose for its growth (v) DEREPRESSION OF lac OPERON is a Double negative control. (vi) Figure below explains (a) repression and (b) derepression conditions of the lac operon. There is repression when the movement/ activity of RNA polymerase is not possible as shown in (a).
  • 7. (vii) Positive control of lac operon : is possible when transcription of the lac operon is stimulated due to CAP/CRP. (viii) lac operon requires the presence of a specific activator protein called catabolite activator protein (CAP), also called cAMP receptor protein (CRP). It is a dimeric protein that first complexes with 35-cyclic AMP (cAMP) and then binds to the lac promoter just upstream from the binding site for RNA polymerase. (ix) This step increases the binding of RNA polymerase and so stimulates the initiation of transcription by approximately a factor of 50. An increase in the cAMP level inside an E. coli bacterium results in the formation of CAP-cAMP complexes. So CAP-cAMP acts as a positive regulator.
  • 8. Watch an animated tutorial about the workings of lac operon here. https://www.openstax.org/l/lac_operon end