Mutations

What is a gene? Prokaryotic Genes PROMOTER 3’ 5’ 3’ (-35) (-10) antisense ---TTGACAT------TATAAT-------AT-/-AGGAGGT-/-ATG CCC CTT TTG TGA ---AACTGTA------ATATTA-------TA-/-TCCTCCA-/-TAC GGG GAA AAC ATT sense 3’ (-35) (-10) RIBOSOME BINDING SITE 5’ 5’ 3’ U-/-AGGAGGU-/-AUG CCC CUU UUG UGA Met Pro leu leu stp When all of these rules are satisfied then A segment of DNA will generate an RNA which will then be read by a ribosome and be translated into a protein.

Reading the genetic code A T G T T T A A A T A G C C C 5’ 3’ C A T A A A T T T C T A G G G 5’ 3’ A T G T T T A A A T A G C C C 5’ 3’ 5’ 3’ A U G U U U A A A U A G C C C 5’ 3’ C A T A A A T T T C T A G G G A U G U U U A A A U A G C C C 5’ 3’ U A C Met A A A Phe U U U Lys S T P

No Gaps A U G U U U A A A U A G C C C 5’ 3’ U A C Met A A A Phe U U U Lys S T P A U G U U U A A A U A G C C C 5’ 3’ U A C Met A A U Asn U U A Leu

No overlaps A U G A A A C C C U A G 5’ 3’ U A C Met U U U Lys G G G Pro S T P A U G A A A C C C U A G 5’ 3’ U A C Met U U U Lys U G G Trp

The GENETIC CODE The code is a three letter code. Second letter U C A UUU UUC UUA UUG CUU CUC CUA CUG AUU AUC AUA AUG GUU GUC GUA GUG UCU UCC UCA UCG CCU CCC CCA CCG ACU ACC ACA ACG GCU GCC GCA GCG UAU UAC UAA UAG CAU CAC CAA CAG AAU AAC AAA AAG GAU GAC GAA GAG UGU UGC UGA UGG CGU CGC CGA CGG AGU AGC AGA AGG GGU GGC GGA GGG U C A G Phe Tyr Cys U Ser STOP STOP Leu Trp His C Arg Pro Leu Gln First letter Third letter Asn Ser Ile A Thr Lys Arg Met Asp G Val Ala Gly Glu

The Genetic Code Properties of the Genetic code: 1- The code is written in a linear form using the nucleotides that comprise the mRNA 2- The code is a triplet: THREE nucleotides specify ONE amino acid 3- The code is degenerate: more than one triplet specifies a given amino acid 4- The code is unambiguous: each triplet specifies only 5- The code contains stop signs- There are three different stops 6- The code is comma less 7- The code is non-overlapping

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Mutations Most mutations are harmful in their effects; only rarely are mutations beneficial. A gene with one wild-type allele is monomorphic; a gene with two or more wild-type alleles is polymorphic. The vast majority of mutant traits are determined by alleles of more than one gene. This means that most traits are multifactorial traits. A Heterogeneous Trait is one that may be caused by mutations in more than one gene. Human deafness is an example of a heterogeneous trait: Mutations in any of at least 50 genes lead to deafness. An important class of mutations are conditional mutations- (Environment affects Phenotype). Conditional mutations are those that express their associated phenotype only under some conditions (restrictive conditions) and not others (permissive conditions). Conditional lethal mutations are common. Temperature-sensitive conditional mutations are invaluable in genetic research.

Generation of mutations Spontaneous mutations Replication induced mutations of DNA Usually base substitutions (Most errors are corrected) Meiosis- segregation defects or defects during crossing over can induce mutations Small additions and deletions AND Large changes as well Environment induced changes Exposure to physical mutagens - Radioactivity or chemicals Depurination (removal of A or G) Repair results in random substitution during replication Deamination (removal of amino group of base) (nitrous acid) Cytosine--uracil--bp adenine--replication-- Oxidation (oxoG) guanine--oxoguanine--bp adenine--replication -- Base analog incorporation during replication BU-T Intercalating agents

Methods used to study mutations Gross chromosomal changes- deletions, insertions, inversions, translocations Cytology- microscopy- karyotype Small mutations Small deletions, insertions and point mutations Recombinant DNA technologies

Missense mutations UUU UUU UGC UUU UUU WT Phe Phe Cys Phe Phe Missense mutations alters ONE codon so that it encodes a different amino acid UUU UUU UGC UUU UUU WT Phe Phe Cys Phe Phe UUU UUU UGG UUU UUU mut Phe Phe Trp Phe Phe

Consequences of Missense Mutations Missense mutations alter one of the many amino acids that make a protein Its consequences depend on which amino acid is altered Conservative mutations: K to R Nonconservative mutations: K to E Surface Vs buried Mutations in globular domains Vs unstructured tails Silent mutations Mutations in non-coding regions Nonsense mutations

NNN UGG CGA UGG CGA UGG CGA UGG CGA NNN Frameshift mutations A single base-pair deletion or insertion results in a change in the reading frame NNN UGG CGA UGG CGA UGG CGA UGG CGA NNN Trp Arg Trp Arg Trp Arg Trp Arg Delete A NNN UGG CGU GGC GAU GGC GAU GGC GAN NN Trp Arg Gly Asp Gly Asp Gly Insert A NNN UGG CGA AUG GCG AUG GCG AUG GCG ANNN Trp Arg Met Ala Met Ala Met Ala

NNN UGGCGAUGGCGAUGGCGAUGGCGA NNN ... TrpArgTrpArgTRpArgTrpArg … Frameshift mutations A single base-pair deletion or insertion results in a change in the reading frame NNN UGGCGAUGGCGAUGGCGAUGGCGA NNN ... TrpArgTrpArgTRpArgTrpArg … .... GlyAspGlyAspGlyAspGlyAsp ..... AlaMetAlaMetAlaMetAla Delete A UGG CGU GGC GAU GGC GAU GGC GANN Trp Arg Gly Asp Gly Asp Gly Insert A UGG ACG AUG GCG AUG GCG AUG GCG ANN Trp Thr Met Ala Met Ala Met Ala

Frameshift mutations- Deletion Base-pair deletion or insertion results in a change in the reading frame AUG UUU AGC UUU AGC UUU AGC Met Phe Ser Phe Ser Phe Ser Delete C AUG UUU AGU UUA GCU UUA GC Met Phe Ser Leu Ala Leu Delete GC AUG UUU AUU UAG CUU UAG C Met Phe Ile Stp Delete AGC AUG UUU UUU AGC UUU AGC Met Phe Phe Ser Phe Ser

Frameshift mutations-Insertion Base-pair deletion or insertion results in a change in the reading frame AUG UUU AGC UUU AGC UUU AGC Met Phe Ser Phe Ser Phe Ser Insert C AUG UUU AGC CUU UAG CUU UAG C Met Phe Ser Leu STOP Insert CC AUG UUU AGC CCU UUA GCU UUA GC Met Phe Ser Pro Leu Ala Leu Insert CCC AUG UUU AGC CCC UUU AGC UUU AGC Met Phe Ser Pro Phe Ser Phe Ser

AUG UUU AGC UUU AGC UUU AGC WT Met Phe Ser Phe Ser Phe Ser Silent Mutations Silent mutations do not alter the amino acid sequence! The Genetic code is degenerate! AUG UUU AGC UUU AGC UUU AGC WT Met Phe Ser Phe Ser Phe Ser AUG UUC AGC UUU AGC UUU AGC Mut Mutations that occur within introns are often silent but mutations at splicing junctions have effects Mutations that occur in non-genic regions are often silent except when they occur in regulatory sequences

Mutations in non-protein coding regions Mutations in the promoter, splicing junction or ribosome binding site are also mutagenic Reduced expression of mRNA might result in reduced levels of proteins OR Increased expression of mRNA might result in increased levels of protein Mutations in splicing junctions may also be mutagenic improperly spliced mRNA will result in the intron being translated Mutations in tRNA or aminoacyl-tRNA synthase are mutagenic

UUU UUU UGC UUU UUU Phe Phe Cys Phe Phe UUU UUU UGA UUU UUU Nonsense mutations Nonsense mutations alter one codon so that it now encodes for a STOP codon UUU UUU UGC UUU UUU Phe Phe Cys Phe Phe UUU UUU UGA UUU UUU Phe Phe STOP Nonsense mutations insert a stop codon which results in premature termination Truncated polypeptide usually results in loss of function for polypeptide

A U G U A C A A A U A G C C C 5’ 3’ U A C Met A U G Trp U U U Lys There are NO tRNAs in wildtype cells with anti-codons that recognize STOP codons in mRNA What happens if there is a mutation in the anti-codon loop of a specific tRNA Gene that allows a tRNA to recognize a stop codon A U G U A C A A A U A G C C C 5’ 3’ U A C Met A U G Trp U U U Lys S T P

Nonsense suppressor mutations! These are the result of a mutation in the anti-codon loop of a specific tRNA Gene It allows the tRNA to recognize a nonsense codon and base pair with it. DNA Gene encoding tRNATRP Point mutation occurs in the anticodon loop OF THE tRNA This allows this tRNA to base pair with a stop codon and ? AUG Trp AUG Trp AUC Trp 5---UAC---UAG---UAA3’ 5--UAC---UAG---UAA3’ Normal tRNA Mutant tRNA

Nonsense suppressor --- UUU UUU UAG UUU UUU ----- --- Phe Phe STP Trp-tRNA has mutation in anticodon. This allows it to pair with a stop codon 5’--- UUU UUU UAG UUU UUU UAA-----3’ AUC Trp AAA Met Ala Phe --- Phe Phe Trp Phe Phe | A mutant protein that is larger than normal will be synthesized!!

Nonsense and Nonsense suppressor --- UUU UUU CAG UUU UUU ----- --- Phe Phe Gln Phe Phe --- Nonsense mutation --- UUU UUU UAG UUU UUU ----- --- Phe Phe STOP AUC Trp ---UAG--- What will happen if an individual carries both a nonsense mutation in a gene and a nonsense suppressor mutation in the anticodon loop of one of the trp-tRNA genes. 5’--- UUU UUU UAG UUU UUU -----3’ AUC Trp AAA Met Ala Phe Phe Phe Trp Phe Phe

Methods used to study mutations Gross chromosomal changes- deletions, insertions, inversions, translocations Cytology- microscopy- karyotype Small mutations Small deletions, insertions and point mutations Recombinant DNA technologies