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Structure & concept of gene, One gene one enzyme hypothesis, Genetic Code PROTEIN SYNTHESIS, Regulation of gene expression Dr. Madhumita Bhattacharjee Assiatant Professor Botany Deptt. P.G.G.C.G. -11,Chandigarh Definitions of the gene • The gene is the unit of genetic information that controls a specific aspect of the phenotype. • The gene is the unit of genetic information that specifies the synthesis of one polypeptide. 1942: George Beadle and Edward Tatum Studied relationships between genes and enzymes in the haploid fungus Neurospora crassa (orange bread mold). Discovered that genes act by regulating definite chemical events. One Gene-One Enzyme Hypothesis Each gene controls synthesis/activity of a single enzyme. “one gene-one polypeptide” 1958: George Beadle (Cal Tech) & Edward Tatum (Rockefeller Institute) Beadle and Tatum (1942)--One Gene, One Enzyme • • • Bread mold Neurospora can normally grow on minimal media, because it can synthesize most essential metabolites. If this biosynthesis is under genetic control, then mutants in those genes would require additional metabolites in their media. This was tested by irradiating Neurospora spores and screening the cells they produced for additional nutritional requirements (auxotrophs). Beadle and Tatum proposed: “One Gene-One Enzyme Hypothesis” However, it quickly became apparent that… 1. More than one gene can control each step in a pathway (enzymes can be composed of two or more polypeptide chains, each coded by a separate gene). 2. Many biochemical pathways are branched. “One Gene-One Enzyme Hypothesis” “One Gene-One Polypeptide Hypothesis” Modern Concept of Gene • Until 1940, the gene was considered as the basic unit of genetic information as defined by three criteria. - Cistron:The unit of function, controlling the inheritance of one “character” or phenotypic attribute. – Recon : The unit of recombination – Muton:The unit of mutation. Genetic code: Def. Genetic code is the nucleotide base sequence on DNA ( and subsequently on mRNA by transcription) which will be translated into a sequence of amino acids of the protein to be synthesized. The code is composed of codons Codon is composed of 3 bases ( e.g. ACG or UAG). Each codon is translated into one amino acid. The 4 nucleotide bases (A,G,C and U) in mRNA are used to produce the three base codons. There are therefore, 64 codons code for the 20 amino acids, and since each codon code for only one amino acids this means that, there are more than one cone for the same amino acid. How to translate a codon (see table): This table or dictionary can be used to translate any codon sequence. Each triplet is read from 5′ → 3′ direction so the first base is 5′ base, 7 followed by the middle base then the last base which is 3′ base. Examples: 5′- A UG- 3′ codes for methionine 5′- UCU- 3′ codes for serine 5′ - CCA- 3′ codes for proline Termination (stop or nonsense) codons: Three of the 64 codons; UAA, UAG, UGA do not code for any amino acid. They are termination codes which when one of them appear in mRNA sequence, it indicates finishing of protein synthesis. Characters of the genetic code: 1- Specificity: the genetic code is specific, that is a specific codon always code for the same amino acid. 2- Universality: the genetic code is universal, that is, the same codon is used in all living organisms, procaryotics and eucaryotics. 3- Degeneracy: the genetic code is degenerate i.e. although each codon corresponds to a single amino acid,one amino acid may have more than one codons. e.g arginine has 6 different codons 8 9 Gene mutation (altering the nucleotide sequence): 1- Point mutation: changing in a single nucleotide base on the mRNA can lead to any of the following 3 results: i- Silent mutation: i.e. the codon containg the changed base may code for the same amino acid. For example, in serine codon UCA, if A is changed to U giving the codon UCU, it still code for serine. See table. ii- Missense mutation: the codon containing the changed base may code for a different amino acid. For example, if the serine codon UCA is changed to be CCA ( U is replaced by C), it will code for proline not serine leading to insertion of incorrect amino acid into polypeptide chain. iii- Non sense mutation: the codon containing the changed base may become a termination codon. For example, serine codon UCA becomes UAA if C is changed to A. UAA is a stop codon leading to termination 10 of translation at that point. How your cell makes very important proteins • The production (synthesis) of proteins. • 3 phases: 1. Transcription 2. RNA processing 3. Translation • DNA RNA Protein DNA RNA Protein Nuclear membrane DNA Transcription Eukaryotic Cell Pre-mRNA RNA Processing mRNA Ribosome Translation Protein Before making proteins, Your cell must first make RNA • How does RNA (ribonucleic acid) differ from DNA (deoxyribonucleic acid)? RNA differs from DNA 1. RNA has a sugar ribose DNA has a sugar deoxyribose 2. RNA contains uracil (U) DNA has thymine (T) 3. RNA molecule is single-stranded DNA is double-stranded 1. Transcription • Then moves along one of the DNA strands and links RNA nucleotides together. Nuclear membrane DNA Transcription Eukaryotic Cell Pre-mRNA RNA Processing mRNA Ribosome Translation Protein 1. Transcription OR RNA production • RNA molecules are produced by copying part of DNA into a complementary sequence of RNA • This process is started and controlled by an enzyme called RNA polymerase. 1. Transcription DNA RNA Polymerase pre-mRNA Types of RNA • Three types of RNA: A. messenger RNA (mRNA) B. transfer RNA (tRNA) C. ribosome RNA (rRNA) • All types of RNAproduced in the nucleus! mRNA • Carries instructions from DNA to the ribosome. • Tells the ribosome what kind of protein to make A. Messenger RNA (mRNA) start codon mRNA A U G G G C U C C A U C G G C G C A U A A codon 1 protein methionine codon 2 codon 3 glycine serine codon 4 isoleucine codon 5 codon 6 glycine alanine codon 7 stop codon Primary structure of a protein aa1 aa2 aa3 peptide bonds aa4 aa5 aa6 rRNA • Part of the structure of a ribosome • Helps in protein production tRNA • Bring right amino acid to make the right protein according to mRNA instructions B. Transfer RNA (tRNA) amino acid attachment site methionine U A C anticodon amino acid RNA Processing Nuclear membrane DNA Transcription Eukaryotic Cell Pre-mRNA RNA Processing mRNA Ribosome Translation Protein RNA Processing (Post Transcriptional Changes) • Introns are pulled out and exons come together. • End product is a mature RNA molecule that leaves the nucleus & move to the cytoplasm. RNA Splicing pre-RNA molecule exon intron exon intron exon intron intron exon splicesome exon exon splicesome exon exon exo n Mature RNA molecule Ribosomes Large subunit P Site A Site mRNA A U G Small subunit C U A C U U C G 3. Translation - making proteins Nuclear membrane DNA Transcription Eukaryotic Cell Pre-mRNA RNA Processing mRNA Ribosome Translation Protein 3. Translation • Three parts: 1. initiation: start codon (AUG) 2. elongation: 3. termination: stop codon (UAG) 3. Translation Large subunit P Site A Site mRNA A U G Small subunit C U A C U U C G Initiation aa1 aa2 2-tRNA 1-tRNA anticodon hydrogen bonds U A C A U G codon G A U C U A C U U C G A mRNA Elongation peptide bond aa3 aa1 aa2 3-tRNA 1-tRNA anticodon hydrogen bonds U A C A U G codon 2-tRNA G A A G A U C U A C U U C G A mRNA aa1 peptide bond aa3 aa2 1-tRNA 3-tRNA U A C (leaves) 2-tRNA A U G G A A G A U C U A C U U C G A mRNA Ribosomes move over one codon aa1 peptide bonds aa4 aa2 aa3 4-tRNA 2-tRNA A U G 3-tRNA G C U G A U G A A C U A C U U C G A A C U mRNA aa1 peptide bonds aa4 aa2 aa3 2-tRNA 4-tRNA G A U (leaves) 3-tRNA A U G G C U G A A C U A C U U C G A A C U mRNA Ribosomes move over one codon aa1 peptide bonds aa5 aa2 aa3 aa4 5-tRNA U G A 3-tRNA 4-tRNA G A A G C U G C U A C U U C G A A C U mRNA peptide bonds aa1 aa5 aa2 aa3 aa4 5-tRNA U G A 3-tRNA G A A 4-tRNA G C U G C U A C U U C G A A C U mRNA Ribosomes move over one codon aa4 aa5 Termination aa199 aa3 primary structure aa2 of a protein aa200 aa1 200-tRNA A C U mRNA terminator or stop codon C A U G U U U A G 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. aa2 aa1 aa3 aa4 aa5 aa199 aa200 Regulation of Gene Expression The control of gene expression • Each cell in the human contains all the genetic material for the growth and development of a human • Some of these genes will be need to be expressed all the time called Constitutive genes • These are the genes that are involved in of vital biochemical processes such as respiration • Other genes are not expressed all the time • They are switched on an off at need called Nonconstitutive genes Operons • An operon is a group of genes that are transcribed at the same time. • They usually control an important biochemical process. Jacob, Monod & Lwoff Inducible Genes - Operon Model • Definition: Genes whose expression is turned on by the presence of some substance – Lactose induces expression of the lac genes • Catabolic pathways Lactose Operon • Structural genes – lac z, lac y, & lac a – P-Promoter – Polycistronic mRNA • R-Regulatory gene – Repressor • Operator • Inducer - lactose Regulatory Gene i Operon p o z y a DNA m-RNA Protein -Galactosidase Transacetylase Permease Lactose Operon • Inducer -lactose Absence of lactose i p y a No lac mRNA • Active repressor • No expression • Inactivation of repressor • Expression z Active – Absence – Presence o Presence of lactose i p o z y a Inactive -GalactosidasePermease Transacetylase 1. When lactose is absent • A repressor protein is continuously synthesised. It sits on a sequence of DNA just in front of the lac operon, the Operator site • The repressor protein blocks the Promoter site where the RNA polymerase settles before it starts transcribing Repressor protein DNA I O Regulator gene Operator site © 2007 Paul Billiet ODWS RNA polymerase Blocked z y lac operon a 2. When lactose is present • A small amount of a sugar allolactose is formed within the bacterial cell. This fits onto the repressor protein at another active site (allosteric site) • This causes the repressor protein to change its shape (a conformational change). It can no longer sit on the operator site. RNA polymerase can now reach its promoter site DNA I O z y Promotor site a Repressible Genes - Operon Model • Definition: Genes whose expression is turned off by the presence of some substance (co-repressor) – Tryptophan represses the trp genes • Co-repressor is typically the end product of the pathway Tryptophan Operon • Structural genes – trp E, trpD, trpC trpB & trpA – Common promoter • Regulatory Gene – Apo-Repressor Regulatory Gene R Operon P O L E D C • Inactive • Operator • Co-repressor – Tryptophan Inactive repressor (apo-repressor) 5 Proteins B A Tryptophan Operon Absence of Tryptophan • Co-repressor -tryptophan – Absence of tryptophan • Gene expression R O L E Inactive repressor (apo-repressor) C B A B A 5 Proteins R P O L E • Activates repressor – No gene expression D Presence of Tryptophan • Negative control – Presence of tryptophan P D C No trp mRNA Inactive repressor (apo-repressor) Trp (co-repressor)