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Genes and How They Work Chapter 15 The Nature of Genes • Early ideas to explain how genes work came from studying human diseases. • Archibald Garrod studied alkaptonuria, 1902 – Garrod recognized that the disease is inherited via a recessive allele – Garrod proposed that patients with the disease lacked a particular enzyme • These ideas connected genes to enzymes. 2 The Nature of Genes • Evidence for the function of genes came from studying fungus. • George Beadle and Edward Tatum, 1941 – studied Neurospora crassa – used X-rays to damage the DNA in cells of Neurospora – looked for cells with a new (mutant) phenotype caused by the damaged DNA 3 The Nature of Genes • Beadle and Tatum looked for fungal cells lacking specific enzymes. – The enzymes were required for the biochemical pathway producing the amino acid arginine. – They identified mutants deficient in each enzyme of the pathway. 4 Fig. 15.1-1 Fig. 15.1-2 Fig. 15.1-3 The Nature of Genes • Beadle and Tatum proposed that each enzyme of the arginine pathway was encoded by a separate gene. • They proposed the one gene – one enzyme hypothesis. • Today we know this as the one gene – one polypeptide hypothesis. 8 The Nature of Genes • The central dogma of molecular biology states that information flows in one direction: DNA RNA protein • Transcription is the flow of information from DNA to RNA. • Translation is the flow of information from RNA to protein. 9 Page 279 11 The Genetic Code • Deciphering the genetic code required determining how 4 nucleotides (A, T, G, C) could encode more than 20 amino acids. • Francis Crick and Sydney Brenner determined that the DNA is read in sets of 3 nucleotides for each amino acid. 12 13 The Genetic Code • Codon: set of 3 nucleotides that specifies a particular amino acid • Reading frame: the series of nucleotides read in sets of 3 (codon) – only 1 reading frame is correct for encoding the correct sequence of amino acids 14 The Genetic Code • Marshall Nirenberg identified the codons that specify each amino acid. • There are 64 possible codons for the 22 amino acids - The genetic code is degenerate • There are also “start” and “stop” codons 15 16 The Genetic Code • Stop codons: 3 codons (UUA, UGA, UAG) in the genetic code used to terminate translation • Start codon: the codon (AUG) used to signify the start of translation • The remainder of the code is degenerate meaning that some amino acids are specified by more than one codon. 17 Gene Expression • Template strand: strand of the DNA double helix used to make RNA • Coding strand: strand of DNA that is complementary to the template strand • RNA polymerase: the enzyme that synthesizes RNA from the DNA template 18 Gene Expression Overview • Transcription proceeds through three steps: – Initiation – RNA polymerase identifies where to begin transcription – Elongation – RNA nucleotides are added to the 3’ end of the new RNA – Termination – RNA polymerase stops transcription when it encounters terminators in the DNA sequence 19 Gene Expression Overview • Translation proceeds through three similar steps: – Initiation – mRNA, tRNA, and ribosome come together – Elongation – tRNAs bring amino acids to the ribosome for incorporation into the polypeptide – Termination – ribosome encounters a stop codon and releases polypeptide 20 Gene Expression Overview • Gene expression requires the participation of multiple types of RNA: - Messenger RNA (mRNA) carries the information from DNA that encodes proteins - Ribosomal RNA (rRNA) is a structural component of the ribosome - Transfer RNA (tRNA) carries amino acids to the ribosome for translation 21 Gene Expression Overview • Gene expression requires the participation of multiple types of RNA: - small nuclear RNA (snRNA) are involved in processing pre-mRNA - signal recognition particle (SRP) is composed of protein and RNA and involved in directing mRNA to the RER - micro-RNA (miRNA) are very small and their role is not clear yet 22 Prokaryotic Transcription • Prokaryotic cells contain a single type of RNA polymerase found in 2 forms: – core polymerase is capable of RNA elongation but not initiation – holoenzyme is composed of the core enzyme and the sigma factor which is required for transcription initiation 23 Page 282 25 Prokaryotic Transcription • A transcriptional unit extends from the promoter to the terminator. • The promoter is composed of – a DNA sequence for the binding of RNA polymerase – the start site (+1) – the first base to be transcribed 26 Prokaryotic Transcription • During elongation, the transcription bubble moves down the DNA template at a rate of 50 nucleotides/sec. • The transcription bubble consists of – RNA polymerase – DNA template – growing RNA transcript 27 28 Prokaryotic Transcription • Transcription stops when the transcription bubble encounters terminator sequences – this often includes a series of A-T base pairs • In prokaryotes, transcription and translation are often coupled – occurring at the same time 29 30 31 Eukaryotic Transcription • RNA polymerase I transcribes rRNA. • RNA polymerase II transcribes mRNA and some snRNA. • RNA polymerase III transcribes tRNA and some other small RNAs. • Each RNA polymerase recognizes its own promoter. 32 Eukaryotic Transcription • Initiation of transcription of mRNA requires a series of transcription factors – transcription factors – proteins that act to bind RNA polymerase to the promoter and initiate transcription 33 Fig. 15.9-1 Fig. 15.9-2 Fig. 15.9-3 Eukaryotic pre-mRNA Splicing • In eukaryotes, the primary transcript must be modified by: – addition of a 5’ cap – addition of a 3’ poly-A tail •The primary transcript must be edited by: – removal of non-coding sequences (introns) – splicing together the coding sequences 37 (exons) Fig. 15.10 Eukaryotic pre-mRNA Splicing • The spliceosome is the organelle responsible for removing introns and splicing exons together. • Small ribonucleoprotein particles (snRNPs) within the spliceosome recognize the intronexon boundaries. – introns – non-coding sequences – exons – sequences that will be translated 39 Fig. 15.11a Fig. 15.11b Fig. 15.11c tRNA and Ribosomes • tRNA molecules carry amino acids to the ribosome for incorporation into a polypeptide – aminoacyl-tRNA synthetases add amino acids to the acceptor arm of tRNA – the anticodon loop contains 3 nucleotides complementary to mRNA codons 43 44 45 46 47 48 tRNA and Ribosomes • The ribosome has multiple tRNA binding sites: – P site – binds the tRNA attached to the growing peptide chain – A site – binds the tRNA carrying the next amino acid – E site – binds the tRNA that carried the last amino acid 49 50 tRNA and Ribosomes • The ribosome has two primary functions: – decode the mRNA – form peptide bonds • Peptidyl transferase is the enzymatic component of the ribosome which forms peptide bonds between amino acids 51 Translation • In prokaryotes, initiation of translation requires the formation of the initiation complex including: – an initiator tRNA charged withNformylmethionine – the small ribosomal subunit – mRNA strand • The ribosome binding sequence of mRNA is complementary to part of rRNA 52 53 Translation • Elongation of translation involves the addition of amino acids: – a charged tRNA binds to the A site if its anticodon is complementary to the codon at the A site – peptidyl transferase forms a peptide bond – the ribosome moves down the mRNA in a 5’ to 3’ direction 54 55 56 Translation • There are fewer tRNAs than codons. - Wobble pairing allows less stringent pairing between the 3’ base of the codon and the 5’ base of the anticodon. • This allows fewer tRNAs to accommodate all codons. 57 Translation • Elongation continues until the ribosome encounters a stop codon. • Stop codons are recognized by release factors which release the polypeptide from the ribosome. 58 59 Translation • In eukaryotes, translation may occur on ribosomes in the cytoplasm or on ribosomes of the RER. - the location depends on the intended destination of the protein • Signal sequences at the beginning of the polypeptide sequence bind to the signal recognition particle (SRP) - the signal sequence and SRP are recognized by RER receptor proteins. 60 Translation • The signal sequence/SRP holds the ribosome on the RER. • As the polypeptide is synthesized it passes through a pore into the interior of the endoplasmic reticulum. 61 62 Fig. 15.22-1 Fig. 15.22-2 Fig. 15.22-3 Table 15.2 Mutation: Altered Genes • Point mutations alter a single base. – base substitution mutations – substitute one base for another • transitions or transversion mutations (missense mutations) – nonsense mutations – create stop codon – frameshift mutations – caused by insertion or deletion of a single base – silent mutations - do not change protein 67 68 69 Page 280-1 Page 280-2 Mutation: Altered Genes • Triplet repeat expansion mutations involve a sequence of 3 DNA nucleotides that are repeated many times 72 Mutation: Altered Genes Chromosomal mutations change the structure of a chromosome. – deletions – part of chromosome is lost – duplication – part of chromosome is copied – inversion – part of chromosome in reverse order – translocation – part of chromosome is moved to a new location 73 74 75 Mutation: Altered Genes • Too much genetic change (mutation) can be harmful to the individual. • Genetic variation (caused by mutation) is necessary for evolutionary change of the species. 76