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In experiments with a 3 base codon system it was shown that the code was a nonoverlapping code, meaning that the bases in one codon were not part of another codon. The presence of 64 codons for 20 amino acids allows for some redundancy, meaning that an amino acid may have more than one codon coding for that amino acid. The fact that the codons for an amino acid were similar lead to the proposal by Crick of the ‘wobble’ hypothesis. In this hypothesis the specificity of the code is more in the first two bases allowing for variation in pairing at the third base without changing the amino acid . 51 example: proline - CCU CCC CCA CCG This system could increase the speed of protein synthesis and reduce errors. Summary - Genetic Code 1) 3 bases per codon 2) non-overlapping code 3) some degeneracy or redundancy in the code 4) can have ‘wobble’ for amino acid specificity with a greater specificity for the first two bases of a codon 5) code is almost universal, a major reason for genetic engineering 52 Conversion of the genetic information into a product requires the translation of the nucleotide sequence in DNA to an amino acid sequence in a protein. This concept of one gene one enzyme was first proposed by Beadle and Tatum (1941) Problem with a direct translation (at least in eukaryotes) DNA is found in the nucleus while protein synthesis occurs in the cytoplasm. An intermediary is needed - Ribonucleic Acid RNA RNA is found in both the nucleus and cytoplasm and is similar to DNA since it also has four bases: A, U, G, C. 53 Translation - The formation of a polypeptide with the amino acid sequence directed by the nucleotide sequence of a specific RNA molecule (mRNA) There are 3 types of RNA needed for translation: messenger RNA (mRNA) transfer RNA (tRNA) ribosomal RNA (rRNA) 54 Messenger RNA large molecular weight (500,000 +) intermediate carrier of the genetic code relatively short-lived but will vary among genes and between prokaryotes and eukaryotes may be translated many times 2 to 10% of cellular RNA amount of modification required prior to translation differs between prokaryotes and eukaryotes 55 Difference in mRNA between eukaryotes and prokaryotes is the processing required after transcription. Eukaryotes Prokaryotes DNA DNA transcription hnRNA mRNA processed in nucleus ready for translation mRNA add cap excised add introns tail ready for translation Eukaryotic gene 56 exon intron exon intron exon exon - region that codes for part of the gene intron- region that does not code for part of the gene How the presence of introns was detected: expected observed mRNA ssDNA mRNA ssDNA Why the need for processing? 57 1) Remove the RNA that does not code for the specific polypeptide. Lead to the idea of split genes - where there are regions within the DNA sequence of a gene that does not appear in the final product. 2) Place a methylated guanine (mG) on the 5’ end of the mRNA. No phosphate mG cap is attached in reverse cap is needed for attachment of the small ribosomal sub-unit during translation. 58 3) Polyadenine tail is added after transcription by a specific adenine polymerase. The poly-A tail may vary in length and may act as an attachment site for proteins that protect or assist in the transport of the mRNA through the cytoplasm. 59 Transfer RNA (tRNA) low molecular weight (25,000 +) 70 to 90 nucleotides in length contains modified bases: examples - inosinic acid hypothxanthine ribothymidylic acid pseudouridine modification of the bases occurs posttranscription. some tRNA require processing. makes up 10-15% of cellular RNA each tRNA is specific for one amino acid there may be more than one tRNA for each amino acid 60 ‘Wobble’ in the anticodon loop of tRNA nucleotide at 5’ end of the anticodon nucleotide at 3’ end of codon G can pair with U or C C can pair with G A can pair with U U can pair with A or G I (inosine) can pair with A, U or C The wobble makes it possible to have only 32 tRNA’s for the 61 possible codons. 61 Ribosomal RNA (rRNA) variable molecular weights variable number of nucleotides (120 to 4800) 70 to 80 percent of the cellular RNA Differs between prokaryotes and eukaryotes in size Prokaryote Eukaryote 70S monosome 80S monosome 50S large 30S small 60S large 40S small 23S rRNA 16S rRNA 28S rRNA 18S rRNA 5S rRNA 5.8S rRNA 5.0S rRNA 31 proteins 21 proteins 50 proteins 33 proteins 62 Translation requires: 61 types of tRNA 20 types of aminoacyl tRNA synthetases Large ribosomal subunit Small ribosomal subunit m RNA modified met - tRNA Initiation, Elongation, and Termination factors 63 Translation Prior to translation Activation of tRNA amino acid + ATP aminoacyl tRNA synthetase amino acid - AMP + specific tRNA aminoacyl tRNA synthetase amino acid - tRNA + AMP 64 Characteristics of Translation 1. mRNA is translated 5’ 3’ 2. Protein synthesis occurs N-terminal C-terminal 3. Leader section of mRNA is not translated 4. Rate of translation 4-15 amino acids/sec 5. Many ribosomes can translate same mRNA (polysome or polysome complex) 6. Modified met-tRNA for first amino acid prokaryotes N-formyl methionine eukaryotes ‘initiator’ methionine 65 Initiation of Translation small ribosomal subunit large ribosomal subunit Mg++ GTP mRNA initiation factors IF1, IF2, and IF3 fmet-tRNA 66 Steps in the initiation of translation: 1) Small subunit attaches to 5’ end of mRNA at the Shine-Dalgarno sequence in the leader sequence assisted by IF3. 2) fmet-tRNA attaches to peptidyl site of the small ribosomal subunit assisted by IF2. 3) Large subunit attaches to the small subunit 67 Elongation of the polypeptide chain 1) Next amino acid - tRNA enters the aminoacyl site assisted by elongation factor EF-Tu 2) Peptide bond formation occurs, catalyzed by peptidyl transferase 3) Release of fmet-tRNA 4) Translocation of the ribosome to the next codon assisted by elongation factor EF-G opening up the aminoacyl site 5) Next amino acid - tRNA enters the aminoacyl site assisted by elongation factor EF-Tu 68 Termination of Translation 1) The ribosome reaches one of three termination codons (UAG, UAA, UGA) 2) Release factor RF1 or RF2 binds to the open aminoacyl site 3) Disassociation occurs resulting in the separation of the following components: mRNA polypeptide small ribosomal subunit large ribosomal subunit final tRNA 69 Translation in Eukaryotes Very similar to prokaryotic translation differences: 5’ cap is the attachment site for the small subunit of the ribosome. Formyl methionine is not required to start translation. Instead a unique initiator tRNAmet is required. The start codon is still AUG. A greater number of initiation, elongation and termination factors are required. 70 Products of Translation polypeptide = protein Classes of proteins enzymes receptor proteins transport proteins structural proteins nucleic acid binding proteins ribosomal proteins storage proteins 71 Protein Structure Structure and function of a protein is controlled by the sequence of the amino acids and the interaction of the amino acids within the polypeptide and with amino acids in other polypeptides. Primary structure: linear sequence of amino acids Secondary structure: interaction of amino acids in the polypeptide in the form of hydrogen bonds that result in the folding of the polypeptide into various shapes/structures. Examples: helix pleated sheets 72 Tertiary structure: additional folding of the polypeptide by covalent bonds forming between amino acid side groups. The folding due to covalent bonds will be more permanent than those found in the secondary structure, why? cyst. S S cyst. Quaternary structure: interaction between polypeptides. Example: enzymes with multiple sub-units 73 Structure of the protein dictates its function. Change the amino acid sequence and you may change the structure of the protein. A change in structure can lead to reduced functionality or non-functionality. So changes in the base sequence of the DNA within a gene can change the functionality of the gene product if the change results in an amino acid(s) change in a critical location of the polypeptide. 74 Gene Mutations Gene mutations are changes in the DNA sequence that cause a detectable change in a gene (i.e. change the expression of a gene). Gene mutations can occur spontaneously or can be induced. There are two basic types of gene mutations: base deletions or additions base change Base deletions or additions cause a frameshift mutation because they change the reading frame of the gene. Add or delete 1 - 2 bases a nonsense mutation Add or delete 3 bases a missense mutation Base changes will cause a missense mutation 75 A single base change can have no impact or a major impact depending on: 1) If the codon changed effects a critical amino acid in the polypeptide 2) if the codon is changed to a stop codon 3) if the codon that is changed is the start codon Base change mutations can either be a forward mutation or a reversion mutation. The forward mutation changes the functional form of the gene to a mutant form. A reversion mutation restores either the original DNA sequence of a gene or restores the function of the gene. Example of this is if the forward mutation was a base deletion (frameshift) a base insertion would be a reversion mutation (restores normal reading frame) 76 Gene mutations can either be spontaneous or are induced. Spontaneous mutations are ones that occur naturally due to an error in replication. A replication error can occur due to a mutation in the proof-reading ability of DNA pol I or III or due to a change in base configuration. A change in the base configuration is called a tautomeric shift where a base goes from the keto to the enol form. For example, if a tautomeric shift occurs in a thymine, it will be paired to guanine instead of adenine. If adenine undergoes a tautomeric shift it will now be read as a guanine and be paired with cytosine instead of thymine. 77 Induced mutations are the result of an artificial factor. Gene mutations can be induced by: sunlight (u.v. light) radiation chemicals Sunlight - u.v. light U.v. light induces mutations by causing adjacent thymines in a DNA strand to connect forming a thymine dimer. --------------------T T ------------------------------------------A A ----------------------At replication the presence of a thymine dimer can cause errors in reading by the DNA polymerase leading to the wrong base(s) being inserted. 78 There are two repair systems for u.v. light induced mutations: light activated repair dark repair The light activated repair requires energy from light to work and is called photoreactivation repair catalyzed by the enzyme photolyase. Dark repair is an excision/repair system where an endonuclease first removes the dimer, DNA pol I replaces the missing bases , and ligase makes the final phosphodiester bond. What happens without a repair system? 79 Humans - xeroderma pigmentosum skin cancer Radiation Radiation can cause damage in two ways. A high energy particle hits a DNA strand removing a base, bases, or whole sections of the DNA causing a break in the DNA. A second way to cause damage is the production of free radicals (molecules lacking an electron) that can react with DNA causing a break in the DNA strand or modifying bases within the strand. The rate of mutation increases with the level of the dose. 80 Chemical mutagens Chemical mutagens will cause a change in a base changing how it will pair in the next cycle of replication. This will cause a change in a codon, i.e. ATT GTT There are two types of base changes, transitions and transversions. Transition: purine purine, ex. AG pyrimidine pyrimidine, ex. CT Transversion: purine pyrimidine, ex. AC - pyrimidine purine, ex. TG Examples of chemical mutagens 81 1. base analogs - where a chemical is similar to a base but pairs differently example 1 - 5-bromouracil (BU) which is an analog to thymine. If you were to go through a series of replications the base change would occur in this way. A A G G T BU BU C So the change is a transition, thymine to cytosine example 2 - aminopurine (AP) an analog to adenine T T A C AP C AP G 82 so the change is a transition, adenine to guanine 2. Deamination - where a chemical will modify the base structure changing pairing in the next cycle of replication. Example - nitrous acid - removes amino groups adenine is changed to hypoxanthine (H) which pairs to cytosine instead of thymine. A H T C amino group G C cytosine is changed to uracil which pairs with adenine instead of guanine. 83 C U G A amino group T A 3. alkylating agents - where a chemical will change the base structure by adding a methyl group, changing base pairing. Example - ethyl methanesulfonate (EMS) will add a methyl group to guanine making it a base analog to thymine. G m G C A methyl group T A 84 Testing for chemical mutagens As new chemicals are developed it is important to determine if they pose a risk to humans/the environment as a mutagen or a carcinogen. What is the difference between a mutagen and a carcinogen? What is the challenge in testing if a chemical is a mutagen? The Ames test was developed to determine if a chemical could induce either frameshift or base change mutations. The test used bacteria that contained either a frameshift mutation or a base change mutation making it dependent on a nutrient supplement in the media for survival (called an auxotroph). 85 If bacteria are detected after treatment with the chemical that do not require the nutrient supplement for survival (become prototroph or wild-type strain) at a higher rate then normal (due to natural errors in replication) the chemical is considered a mutagen. Why is it easier to detect mutations from auxotrophs to prototrophs then prototrophs to auxotrophs? What has been the complaint about testing chemicals in this way? 86