Genetic Code PDF

# Introduction - Genetic Code The letters A, G, T, and C correspond to the nucleotides found in DNA. They are organized into codons. The collection of codons is called Genetic code. For 20 amino acids, there should be 20 codons. Each codon should have 3 nucleotides to impart specificity to each of...

# Introduction - Genetic Code The letters A, G, T, and C correspond to the nucleotides found in DNA. They are organized into codons. The collection of codons is called Genetic code. For 20 amino acids, there should be 20 codons. Each codon should have 3 nucleotides to impart specificity to each of the amino acid for a specific codon. - 1 Nucleotide - 4 combinations - 2 Nucleotides - 16 combinations - 3 Nucleotides - 64 combinations (Most suited for 20 amino acids) # Genetic Code - Genetic code is a dictionary that corresponds with the sequence of nucleotides and the sequence of Amino Acids. - Words in the dictionary are in the form of codons. - Each codon is a triplet of nucleotides - 64 codons in total and three out of these are Non Sense codons. - 61 codons for 20 amino acids. # Introduction - Translation The pathway of protein synthesis is called Translation because the language of the nucleotide sequence on mRNA is translated into the language of an amino acid sequence. The process of Translation requires a Genetic code, through which the information contained in nucleic acid sequence is expressed to produce a specific sequence of amino acids. # Genetic Code - **Salient features of genetic code** - **Universal** - the codons code for any amino acid in any organism, be it a bacterium or a human being. - **Non ambiguous** - each codon codes for only one amino acid, so the genetic code is unambiguous and specific. - **Comma less** - the codons are read in the 5'3' direction, and there is no punctuation. - **Degenerate** - some amino acids are coded by more than one codon, the genetic code is said to be Degenerate. - **Non overlapping** - 3 successive nitrogen bases code for only one amino acid. - **Nonsense codon** (UAA, UAG, UGA) - do not code for any amino acids, but act as terminating/stop codons of protein synthesis. - **Linear** - the sequence of amino acids present in a polypeptide chain corresponds to a sequence of nitrogen bases of DNA with 3 successive nitrogen bases forming a single codon. - **Triplet** - there are 64(4x4x4) codons, 61 codons code for 20 amino acids. - **Initiation Codon AUG** # The Genetic Code | First Base | Second Base | Third Base | Amino Acid | |:----------:|:------------:|:----------:|:---------------------------------------------:| | U | U | U | Phenylalanine | | U | U | C | Phenylalanine | | U | U | A | Tyrosine | | U | U | G | Cysteine | | U | C | U | Serine | | U | C | C | Serine | | U | C | A | Tyrosine | | U | C | G | Stop | | U | A | U | Leucine | | U | A | C | Leucine | | U | A | A | Stop | | U | A | G | Stop | | U | G | U | Leucine | | U | G | C | Leucine | | U | G | A | Stop | | U | G | G | Tryptophan | | C | U | U | Leucine | | C | U | C | Leucine | | C | U | A | Leucine | | C | U | G | Leucine | | C | C | U | Proline | | C | C | C | Proline | | C | C | A | Proline | | C | C | G | Proline | | C | A | U | Histidine | | C | A | C | Histidine | | C | A | A | Glutamine | | C | A | G | Glutamine | | C | G | U | Arginine | | C | G | C | Arginine | | C | G | A | Arginine | | C | G | G | Arginine | | A | U | U | Isoleusine | | A | U | C | Isoleusine | | A | U | A | Isoleusine | | A | U | G | Methionine | | A | C | U | Threonine | | A | C | C | Threonine | | A | C | A | Threonine | | A | C | G | Threonine | | A | A | U | Asparagine | | A | A | C | Asparagine | | A | A | A | Lysine | | A | A | G | Lysine | | A | G | U | Serine | | A | G | C | Serine | | A | G | A | Arginine | | A | G | G | Arginine | | G | U | U | Valine | | G | U | C | Valine | | G | U | A | Valine | | G | U | G | Valine | | G | C | U | Alanine | | G | C | C | Alanine | | G | C | A | Alanine | | G | C | G | Alanine | | G | A | U | Aspartic acid | | G | A | C | Aspartic acid | | G | A | A | Glutamic acid | | G | A | G | Glutamic acid | | G | G | U | Glycine | | G | G | C | Glycine | | G | G | A | Glycine | | G | G | G | Glycine | # Important Features of the Genetic Code - Each codon consists of three bases (triplet). There are 64 codons. They are all written in the 5' to 3' direction. - 61 codons code for amino acids. The other three (UAA, UGA, UAG) are stop codons (or nonsense codons) that terminate translation. - There is one start codon (initiation codon), AUG, coding for methionine. Protein synthesis begins with methionine (Met) in eukaryotes, and formylmethionine (fmet) in prokaryotes. - The code is unambiguous. Each codon specifies no more than one amino acid. # Describe the Characteristics of the Genetic Code - Written in linear form - Each word consists of 3 ribonucleotide letters - The code is unambiguous - The code is degenerate - The code contains 1 start and 3 stop codons - The code is commaless - The code is non-overlapping - The code is (nearly) universal # Characteristic of the Genetic Code - Triplet code - Comma less - Nonoverlapping code - The coding dictionary - Degenerate code - Universality of code - Non ambiguous code - Chain inition code - Chain termination codons # Salient Features of Genetic Code 1. **Triplet codons:** Each codon is a consecutive sequence of three bases on the mRNA, e.g. UUU codes for phenylalanine. 2. **Non overlapping:** The codes are consecutive. Therefore, the starting point is extremely important. The codes are read one after another in a continuous manner, e.g. AUG, CAU, CAU, GCA, etc. 3. **Non Punctuated:** There is no punctuation between the codons. It is consecutive or continuous. 4. **Degenerate:** When an amino acid has more than one codon, it is called degeneracy of the code. E.g. serine has 6 codons, while glycine has 4 codons. 5. **Unambiguous:** Though the codons are degenerate, they are unambiguous: or without any doubtful meaning. That is, one codon codes for only one amino acid. # Redundancy of the Genetic Code Another term for this is degenerate. There are many situations where different codons specify the same amino acid. But no codon will ever specify for two different amino acids. The codons encoding for one amino acid will usually differ in the third or second position. This makes it more difficult for mutations to cause serious issues. # Complementary Base Pairing fMet - 3' end A - C - C - 5' end - Complementary binding - UAC - AUG - Anticodon - 5' - 3' mRNA - Codon # The Wobble Hypothesis UCC and UCU both code for serine. - tRNA anticodon loop - Anticodon - 3' - A - G - G - Wobble position - 5' - 5' - U - C - C/U - Codon - 3' mRNA # Wobble - When several codons encode the same amino acid, the difference is usually in the third position. - If an anticodon recognizes a codon as a triplet, there should be tRNAs for each codon. - But some anticodons of tRNAs contain inosinate (I) - It forms rather weak bonds than Watson-Crick base pairs. - Anticodon - 3 2 1 - G - C - I - A - U - C - Codon - 5' C - G - A - 1 2 3 - 3 2 1 - G - C - I - C - G - U - 1 2 3 - 3 2 1 - G - C - I (5') - C - G - C (3') - 1 2 3 # Wobble and Normal Pairing - Leu - 3' - 5' - Identical leucine tRNAs - Leu - 3' - 5' - GAG - C - U - C - Normal pairing - mRNA - 5'... - ... - 3' - GAG - C - U - U - Wobble pairing - mRNA - 5'... - ... - 3' # Universal The codons are the same for the same amino acid in all species; the same for "Elephant and E.coli". The genetic code has been highly preserved during evolution. # Terminator Codons There are three codons which do not code for any particular amino acids. They are "nonsense codons", more correctly termed as punctuator codons or terminator codons. They put "full stop" to the protein synthesis. These three codons are UAA, UAG, and UGA. # Initiator Codon In most of the cases, AUG acts as the initiator codon. # Genetic Code - Universal - **Universal** - In all living organisms, the Genetic code is the same. - The exception to universality is found in mitochondrial codons where AUA codes for methionine and UGA for tryptophan, instead of isoleucine and termination codon respectively of cytoplasmic protein synthesizing machinery. - AGA and AGG code for Arginine in cytoplasm but in mitochondria, they are termination codons. - The universality of the genetic code is among the strongest evidence that **all living things share a common evolutionary heritage**. # Universal In all living organisms, The Genetic code is the same. - The exception to universality is found in mitochondrial codons where: - AUA is methionine - UGA is tryptophan - In Cytoplasmic codons: - AUA is isoleucine - UGA is the termination codon - AGA and AGG code for Arginine in cytoplasm but in mitochondria, they are termination codons. # Marshall Nirenberg and Heinrich Matthaei experiments - The 2nd experiments AAAAAA..... Result: Peptide of lysine - The 3rd experiments CCCCCC.... Result: Peptide for Proline. - GGGGGGG.... was unstable, so this part of the experiment could not be done. - Next to prove other codon-amino acid pairs hence researchers synthesized chains of alternating bases # Genetic Code Cracking Phase 1 - Nirenberg & Matthaei take bacterial cell extract containing ribosomes and other stuff needed to make proteins - all but the instructions. - Thr - Trp - Ser - Arg - Asn - Cys - Pro - Ala - Include a mix of amino acids, where 1 at a time is radiolabeled. - Leu - Lys - Gln - Glu - Asp - Ile - Gly - Val - His - Phe - Met - Tyr - Stick in different RNA sequences to act as instructions - UUUUUUUUUUUUU - CCCCCCCCCCCCC - AAAAAAAAAAAAAAAA - Let protein be made. - Measure radioactivity compared to the amount of protein made. It'll only be high when the amino acid spelled by the sequence is labeled. # Experiment **Question:** What amino acids are specified by codons composed of only one type of base? - **Methods:** - Uracil nucleotides - Polynucleotide phosphorylase - Poly(U) homopolymer - **It's** - A homopolymer - in this case, poly(U) mRNA - was added to a test tube containing a cell-free translation system, 1 radioactively labeled amino acid, and 19 unlabeled amino acids. - The tube was incubated at 37°C. - Translation took place. - The protein was filtered, and the filter was checked for radioactivity. - The procedure was repeated in 20 tubes, with each tube containing a different labeled amino acid. - The tube in which the protein was radioactively labeled contained newly synthesized protein with the amino acid specified by the homopolymer. In this case, UUU specified the amino acid phenylalanine. - **Conclusion:** - UUU codes for phenylalanine; in other experiments, AAA was found to code for alanine, and CCC for proline. - Nirenberg and Matthaei developed a method for identifying the amino acid specified by a homopolymer. # Ribosome - Amino acid chain (protein) - Large subunit - Ribosome - Small subunit - Codon - Ribosome - tRNA - mRNA - Shutterstock.com - 1698956266 # Experiment **Question:** With the use of tRNAs, what other matches between codon and amino acid could be determined? - **Methods:** - Very short mRNAs with known codons were synthesized.... - ...and added to a mixture of ribosomes and tRNAs attached to amino acids. - **Synthetic mRNA with one codon** - GUU - GCA - CUC - **tRNAs with amino acids** - **Mix** - Ribosome - **Urbound tRNAS** - CUC - GCA - CAA - GUU - **Ribosome with mRNA and tRNA specified by codon** - **Filter solution** - **Results** - The ribosome bound the mRNA and the tRNAs that it specified. - The mixture was then passed through a nitrocellulose filter. The tRNAs paired with ribosome-bound mRNA stuck to the filter, whereas unbound tRNAs passed through. - The tRNAs on the filter were bound to valine. - **Conclusion:** - The codon GUU specifies valine; many other codons were determined by using this method. - **Nirenberg and Leder developed a technique for using ribosome-bound tRNAs to provide additional information about the genetic code.** # Experiment **Question:** With the use of tRNAs, what other matches between codon and amino acid could be determined? - **Methods:** - 1 Very short mRNAs with known codons were synthesized. - 2 ...and added to a mixture of ribosomes and tRNAs attached to amino acids. - **GUU** - Synthetic mRNA with one codon - **GCA** - tRNAs with ammo acids - **CUC** - Mix - **Ribosome** - **Unbound tRNAs** - CUC - GCA - **Val** - Ribosome with mRNA and tRNA specified by codon - **Glu** - Filter solution - **CAA** - **GUU** - **Arg** - **Results:** - 3 The ribosome bound the mRNA and the tRNAs specified by the mRNA. - 4 The mixture was then passed through a nitrocellulose filter. The tRNAs paired with ribosome-bound mRNA stuck to the filter, whereas unbound tRNAs passed through it. - 5 The filter was assayed to determine which amino acid was bound. - **Conclusion:** When an mRNA with GUU was added, the tRNAs on the filter were bound to valine; therefore the codon GUU specifies valine. Many other codons were determined by using this method. # Filter - mRNA - Charged tRNA - ribosome - mRNA (triplet) and free charged tRNA pass through filter - Ribosomes cannot pass through filter - Specific tRNA's complex with mRNA/ribosome (specific tRNA bound to filter) # Thank You # Termination of Translation - **(a)** - 5' - Ribosome - mRNA - UCC - AGGUAG - PA - 3' - When the ribosome translocates to a stop codon, there is no tRNA with an anticodon that can pair with the codon in the A site. - Stop codon - RF-1 and RF-3 - Release factors. - **(b)** - Polypeptide - 5' - UCC - AGGUAG - EPA - 3' - RF-1 attaches to the A site. - RF-3 - GTP - RF-1 - The polypeptide is released from the tRNA in the P site. - And RF-3 forms a complex with GTP and binds to the ribosome. - **(c)** - 5' - AGGUAG - 3' - RF-1 - GTP associated with RF-3 is hydrolyzed to GDP. - RF-3 GDP + - The tRNA, mRNA, and release factors are released from the ribosome. # Initiation of Translation - **(a)** - Large subunit (50S) - Ribosome - Small subunit (30S) - **(b)** - Shine-Dalgarno sequence - Initiation codon - AUGUGC - mRNA - IF-3 - The ribosome consists of two subunits. - IF-3 binds to the small subunit, preventing the large subunit from binding. - ... thus allowing the small subunit to attach to mRNA - **(c)** - tRNA - FMet - Anticodon - UAC - 30S initiation complex - FMet - mRNA - UAC - IF-2 - GTP - AUGUGC - IF-1 - A tRNA charged with N-formylmethionine forms a complex with IF-2 and GTP... - ...and binds to the initiation codon while IF-1 joins the small subunit. - **(d)** - 70S initiation complex - mRNA - FMet - UAC - AUGUGC - IF-3 - IF-1 - All initiation factors dissociate from the complex, GTP is hydrolyzed to GDP... - ... and the large subunit joins to create a 70S initiation complex. - Next codon - **Conclusion:** At the end of initiation, the ribosome is assembled on the mRNA, and the first tRNA is attached to the initiation codon. # Nirenberg and Leder Experiment - leu - GAU - UAG - GCU - arg - Amino-acid-charged tRNAs - GAU - leu* - UAG - ile - GCU - AUC - UAG - ile - RNA triplets of known sequence - gly - arg - CUA - GAU - GCU - arg - ile* - GAU - leu - Ribosome - leu* - Filter - arg - GCU - UAG - ile - CCG