Genes and How They Work Chapter 15 The Nature of Genes Early ideas to explain how genes work came fromstudying human diseases Archibald Garrod 1902 Recognized that alkaptonuria is inherited via a recessiveallele Proposed that patients with the disease lacked a particularenzyme These ideas connected genes to enzymes Beadle and Tatum 1941 Deliberately set out to create mutationsin chromosomes and verify that theybehaved in a Mendelian fashion in crosses Studied Neurospora crassa Used X-rays to damage DNA Looked for nutritional mutations Had to have minimal media supplemented to grow Beadle and Tatum looked for fungal cells lacking specific enzymes
The enzymes were required for the biochemical pathwayproducing the amino acid arginine They identified mutants deficient in each enzyme of thepathway One-gene/one-enzyme hypothesis has been modifiedto one-gene/one-polypeptide hypothesis Central Dogma First described by Francis Crick
Information only flows from DNA RNA protein Transcription = DNA RNA Translation = RNA protein Retroviruses violate this order using reversetranscriptase to convert their RNA genome intoDNA Transcription Translation DNA-directed synthesis of RNA
Only template strand of DNA used U (uracil) in DNA replaced by T (thymine) in RNA mRNA used to direct synthesis of polypeptides Translation Synthesis of polypeptides Takes place at ribosome Requires several kinds of RNA RNA All synthesized from DNA template by transcription
Messenger RNA (mRNA) Ribosomal RNA (rRNA) Transfer RNA (tRNA) Small nuclear RNA (snRNA) Signal recognition particle RNA Micro-RNA (miRNA) Genetic Code Francis Crick and Sydney Brenner determined how the order ofnucleotides in DNA encoded amino acid order Codon block of 3 DNA nucleotides corresponding to an amino acid Introduced single nulcleotide insertions or deletions and looked formutations Frameshift mutations Indicates importance of reading frame Marshall Nirenberg identified the codons that specify each amino acid
Stop codons 3 codons (UUA, UGA, UAG) used to terminate translation Start codon Codon (AUG) used to signify the start of translation Code is degenerate, meaning that some amino acidsare specified by more than one codon Code practically universal
Strongest evidence that all living things sharecommon ancestry Advances in genetic engineering Mitochondria and chloroplasts have some differencesin stop signals Prokaryotic transcription
Single RNA polymerase Initiation of mRNA synthesis does not requirea primer Requires Promoter Start siteTranscription unit Termination site Promoter Forms a recognition and binding site for the RNA polymerase
Found upstream of the start site Not transcribed Asymmetrical indicate site of initiation anddirection of transcription 15 TATAAT Promoter (10 sequence) Holoenzyme Core enzyme
Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. TATAAT Promoter (10 sequence) Holoenzyme Core enzyme Downstream 5 9 3 Start site (+1) Template strand TTGACAPromoter (35 sequence) Coding strand Prokaryotic RNA polymerase Upstream 5 3 a. b. binds to DNA RNApolymerase bound to unwound DNA Transcription bubble 5 3 dissociates ATP Helix opens at 10 sequence Start site RNA synthesis begins 5 3 15 Elongation Grows in the 5-to-3 direction as ribonucleotidesare added Transcription bubble contains RNA polymerase,DNA template, and growing RNA transcript After the transcription bubble passes, the now- transcribed DNA is rewound as it leaves the bubble Termination Marked by sequence that signals stop to polymerase
Causes the formation of phosphodiester bonds to cease RNADNA hybrid within the transcription bubble dissociates RNA polymerase releases the DNA DNA rewinds Hairpin Prokaryotic transcription is coupled to translation
mRNA begins to be translated before transcriptionis finished Operon Grouping of functionally related genes Multiple enzymes for a pathway Can be regulated together Eukaryotic Transcription
3 different RNA polymerases RNA polymerase I transcribes rRNA RNA polymerase II transcribes mRNA and somesnRNA RNA polymerase III transcribes tRNA and someother small RNAs Each RNA polymerase recognizes its ownpromoter Initiation of transcription
Requires a series of transcription factors Necessary to get the RNA polymerase II enzyme to a promoter and to initiate gene expression Interact with RNA polymerase to form initiation complex at promoter Termination Termination sites not as well defined 24 Other transcription factors RNA polymerase II Eukaryotic DNA
Initiation complex TATAbox 24 mRNA modifications In eukaryotes, the primary transcript must bemodified to become mature mRNA Addition of a 5 cap Protects from degradation; involved in translation initiation Addition of a 3 poly-A tail Created by poly-A polymerase; protection from degradation Removal of non-coding sequences (introns) Pre-mRNA splicing done by spliceosome 5 cap HO OH P P P CH2 + 3 poly-A tail 3 N+ A A A A A A A CH3
Methyl group A A U A A A mRNA P P P G 5 CH3 Eukaryotic pre-mRNA splicing
Introns non-coding sequences Exons sequences that will be translated Small ribonucleoprotein particles (snRNPs)recognize the intronexon boundaries snRNPs cluster with other proteins to formspliceosome Responsible for removing introns Eukaryotic pre-mRNA splicing
Introns non-coding sequences Exons sequences that will be translated Small ribonucleoprotein particles (snRNPs)recognize the intronexon boundaries snRNPs cluster with other proteins to formspliceosome Responsible for removing introns E1 I1 E2 I2 E3 I3 E4 I4 Transcription Introns are removed a. Exons Introns Mature mRNA 33 poly-A tail 5 cap 3 poly-A tail Primary RNA transcript DNAtemplate Alternative splicing Single primary transcript can be spliced into differentmRNAs by the inclusion of different sets of exons 15% of known human genetic disorders are due toaltered splicing 35 to 59% of human genes exhibit some form ofalternative splicing Explains how 25,000 genes of the human genomecan encode the more than 80,000 different mRNAs tRNA and Ribosomes tRNA molecules carry amino acids to theribosome for incorporation into a polypeptide Aminoacyl-tRNA synthetases add amino acids tothe acceptor stem of tRNA Anticodon loop contains 3 nucleotidescomplementary to mRNA codons 2D Cloverleaf Model Acceptor end Anticodon loop 3 5 3D Ribbon-like Model Acceptor end Anticodon loop Icon Anticodon end Acceptor end tRNA charging reaction
Each aminoacyl-tRNA synthetase recognizes only 1amino acid but several tRNAs Charged tRNA has an amino acid added using theenergy from ATP Can undergo peptide bond formation without additionalenergy Ribosomes do not verify amino acid attached to tRNA The ribosome has multiple tRNA binding sites
P site binds the tRNA attached to thegrowing peptide chain A site binds the tRNA carrying the nextamino acid E site binds the tRNA that carried the lastamino acid mRNA 3 5 Large subunit Small The ribosome has two primary functions
Decode the mRNA Form peptide bonds Peptidyl transferase Enzymatic component of the ribosome Forms peptide bonds between amino acids Translation In prokaryotes, initiation complex includes
Initiator tRNA charged with N-formylmethionine Small ribosomal subunit mRNA strand Ribosome binding sequence (RBS) of mRNApositions small subunit correctly Large subunit now added Initiator tRNA bound to P site with A siteempty Initiations in eukaryotes similar except
Initiating amino acid is methionine More complicated initiation complex Lack of an RBS small subunit binds to 5 cap ofmRNA Elongation adds amino acids
2nd charged tRNA can bind to empty A site Requires elongation factor called EF-Tu to bind totRNA and GTP Peptide bond can then form Addition of successive amino acids occurs as acycle There are fewer tRNAs than codons
Wobble pairing allows less stringent pairingbetween the 3 base of the codon and the 5base of the anticodon This allows fewer tRNAs to accommodate allcodons Termination Elongation continues until the ribosome encountersa stop codon Stop codons are recognized by release factorswhich release the polypeptide from the ribosome Protein targeting In eukaryotes, translation may occur in the cytoplasm orthe rough endoplasmic reticulum (RER) Signal sequences at the beginning of the polypeptidesequence bind to the signal recognition particle (SRP) The signal sequence and SRP are recognized by RERreceptor proteins Docking holds ribosome to RER Beginning of the protein-trafficking pathway Copyright The McGraw-Hill Companies, Inc
Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. RNA polymerase II RNA polymerase II 1. RNA polymerase II in the nucleus copies one strand of the DNA to produce the primary transcript. 3 3 Primary RNA transcript Primary RNA transcript 2. The primary transcript is processed by addition of a 5 methyl-G cap, cleavage and polyadenylation of the 3 end, and removal of introns. The mature mRNA is then exported through nuclear pores to the cytoplasm. Poly-A tail Poly-A tail 5 5 Primary RNA transcript Primary RNA transcript Cut intron Cut intron Mature mRNA Mature mRNA 5 cap 5 cap 3. The 5 cap of the mRNA associates with the small subunit of the ribosome. The initiator tRNA and large subunit are added to form an initiation complex. Large subunit 5 cap mRNA Small subunit Cytoplasm Cytoplasm Empty tRNA moves into E site and is ejected Lengthening polypeptide chain Amino acids tRNA arrivesin A site 3 Emptyt RNA 3 3 mRNA A site P site 5 E site 5 5 4. The ribosome cycle begins with the growing peptide attached to the tRNA in the P site. The next charged tRNA binds to the A site with its anticodon complementary to the codon in the mRNA in this site. 5. Peptide bonds form between the amino terminus of the next amino acid and the carboxyl terminus of the growing peptide. This transfers the growing peptide to the tRNA in the A site, leaving the tRNA in the P site empty. 6. Ribosome translocation moves the ribosome relative to the mRNA and its bound tRNAs. This moves the growing chain into the P site, leaving the empty tRNA in the E site and the A site ready to bind the next charged tRNA. 42 Mutation: Altered Genes
Point mutations alter a single base Base substitution substitute one base foranother Silent mutation same amino acid inserted Missense mutation changes amino acid inserted Transitions Transversions Nonsense mutations changed to stop codon Nonpolar (hydrophobic)
Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Normal HBB Sequence Normal Deoxygenated Tetramer Abnormal Deoxygenated Tetramer Polar Leu Thr Pro Glu Glu Lys Ser Amino acids 1 2 1 2 1 2 1 2 C T G A C T C C T G A G A A G A A G T C T Nucleotides Hemoglobin tetramer "Sticky" non- polar sites Abormal HBB Sequence Nonpolar (hydrophobic) Leu Thr Pro val Glu Lys Ser Amino acids Tetramers form long chains when deoxygenated. This distorts the normal red blood cell shape into a sickle shape. C T G A C T C C T G T G A A G A A G T C T Nucleotides 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 anew location Mutations are the starting point for evolution
Too much change, however, is harmful to theindividual with a greatly altered genome Balance must exist between amount of newvariation and health of species