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The structure of DNA Deoxyribonucleic acid DNA is made of nucleotides Each nucleotide is composed of phosphate, sugar (deoxyribose) and a nitrogen base 4 nitrogen bases – Adenine, Thymine, Guanine, Cytosine (A,T,G,C) A-T, C-G Bases are linked by hydrogen bonds Figure 16.5 Sugar–phosphate backbone Nitrogenous bases 5 end Thymine (T) Adenine (A) Cytosine (C) Phosphate Guanine (G) Sugar (deoxyribose) DNA nucleotide 3 end Nitrogenous base Figure 16.7 C 5 end G C Hydrogen bond G C G C G 3 end A T 3.4 nm A T C G C G A T 1 nm C A G C G A G A T 3 end T A T G C T C C G T A (a) Key features of DNA structure 0.34 nm 5 end (b) Partial chemical structure (c) Space-filling model Base Pairing DNA strands Run in opposite directions Helicase – unwinds helix Topoisomerase – cuts and rejoins the helix Ligase – brings together Okazaki fragments DNA polymerases add nucleotides only to the free 3end of a growing strand; therefore, a new DNA strand can elongate only in the 5to 3direction Along one template strand of DNA, the DNA polymerase synthesizes a leading strand continuously, moving toward the replication fork To elongate the other new strand, called the lagging strand, DNA polymerase must work in the direction away from the replication fork The lagging strand is synthesized as a series of segments called Okazaki fragments, which are joined together by DNA ligase Figure 16.13 Primase 3 Topoisomerase 3 5 RNA primer 5 3 Helicase 5 Single-strand binding proteins Figure 16.15b Origin of replication 3 5 RNA primer 5 3 3 Sliding clamp DNA pol III Parental DNA 5 3 5 5 3 3 5 Figure 16.17 Overview Origin of replication Leading strand Lagging strand Leading strand Lagging strand Overall directions of replication Leading strand DNA pol III 5 3 3 Parental DNA Primer 5 3 Primase 5 DNA pol III 4 Lagging strand DNA pol I 35 3 2 DNA ligase 1 3 5 Protein Synthesis 1. Transcription The transfer of genetic info. From DNA to messenger RNA (mRNA) 2. Translation The transfer of mRNA to protein Genes are pieces of DNA that code for proteins mRNA – Uracil instead of Thymine Transcription DNA codes for single strand of mRNA This happens in the nucleus RNA polymerase binds to the promoter region on DNA template Sigma factor recognizes binding site on DNA mRNA detatches at the terminator region of the DNA template Transcription Translation The transfer of mRNA into a protein This happens at the ribosome Every 3 base pairs of mRNA is called a codon tRNA hold anti-codons and amino acids tRNA bring amino acids down to the ribosomes using the corresponding anticodon. Translation Translation The Genetic Code Discovery of DNA – Rosalind Franklin Watson and Crick Mutations Sickle cell mutation Prokaryotes vs. Eukaryotes In prokaryotes, translation of mRNA can begin before transcription has finished In a eukaryotic cell, the nuclear envelope separates transcription from translation Eukaryotic RNA transcripts are modified through RNA processing to yield the finished mRNA A primary transcript is the initial RNA transcript from any gene prior to processing Comparing Gene Expression in Bacteria, Archaea, and Eukarya • Bacteria and eukarya differ in their RNA polymerases, termination of transcription, and ribosomes; archaea tend to resemble eukarya in these respects • Bacteria can simultaneously transcribe and translate the same gene • In eukarya, transcription and translation are separated by the nuclear envelope • In archaea, transcription and translation are likely coupled © 2011 Pearson Education, Inc. Figure 17.4 DNA template strand 5 3 A C C A A A C T T T G G T C G A G G G C T T C A 3 5 DNA molecule Gene 1 TRANSCRIPTION Gene 2 U G G mRNA U U U G G C U C A 5 3 Codon TRANSLATION Protein Trp Phe Gly Ser Gene 3 Amino acid Important vocabulary in transcription The stretch of DNA that is transcribed is called a transcription unit Transcription factors (sigma) – initiate the binding of the RNA polymerase The completed assembly of transcription factors and RNA polymerase II bound to a promoter is called a transcription initiation complex A promoter called a TATA box is crucial in forming the initiation complex in eukaryotes Figure 17.8 1 A eukaryotic promoter Promoter Nontemplate strand DNA 5 3 3 5 T A T A A AA A T AT T T T TATA box Transcription factors Start point Template strand 2 Several transcription factors bind to DNA 5 3 3 5 3 Transcription initiation complex forms RNA polymerase II Transcription factors 5 3 5 3 RNA transcript Transcription initiation complex 3 5 RNA processing • Enzymes in the eukaryotic nucleus modify pre-mRNA (RNA processing) before the genetic messages are dispatched to the cytoplasm • During RNA processing, both ends of the primary transcript are usually altered • Also, usually some interior parts of the molecule are cut out, and the other parts spliced together • Each end of a pre-mRNA molecule is modified in a particular way – The 5 end receives a modified nucleotide 5 cap – The 3 end gets a poly-A tail • These modifications share several functions – They seem to facilitate the export of mRNA to the cytoplasm – They protect mRNA from hydrolytic enzymes – They help ribosomes attach to the 5 end Figure 17.10 5 G Protein-coding segment P P P 5 Cap 5 UTR Polyadenylation signal AAUAAA Start codon Stop codon 3 UTR 3 AAA … AAA Poly-A tail RNA Splicing • In some cases, RNA splicing is carried out by spliceosomes • Spliceosomes consist of a variety of proteins and several small nuclear ribonucleoproteins (snRNPs) that recognize the splice sites Figure 17.12-3 RNA transcript (pre-mRNA) 5 Exon 1 Intron Protein snRNA Exon 2 Other proteins snRNPs Spliceosome 5 Spliceosome components 5 mRNA Exon 1 Exon 2 Cut-out intron Figure 17.13 Gene DNA Exon 1 Intron Exon 2 Intron Exon 3 Transcription RNA processing Translation Domain 3 Domain 2 Domain 1 Polypeptide Figure 17.15 3 Amino acid attachment site 5 Amino acid attachment site 5 3 Hydrogen bonds Hydrogen bonds A A G 3 Anticodon (a) Two-dimensional structure Anticodon (b) Three-dimensional structure 5 Anticodon (c) Symbol used in this book Figure 17.22 1 Ribosome 5 4 mRNA Signal peptide 3 SRP 2 ER LUMEN SRP receptor protein Translocation complex Signal peptide removed ER membrane Protein 6 CYTOSOL What Is a Gene? Revisiting the Question • The idea of the gene has evolved through the history of genetics • We have considered a gene as – A discrete unit of inheritance – A region of specific nucleotide sequence in a chromosome – A DNA sequence that codes for a specific polypeptide chain © 2011 Pearson Education, Inc. Figure 17.26 DNA TRANSCRIPTION 3 5 RNA polymerase RNA transcript Exon RNA PROCESSING RNA transcript (pre-mRNA) AminoacyltRNA synthetase Intron NUCLEUS Amino acid AMINO ACID ACTIVATION tRNA CYTOPLASM mRNA Growing polypeptide 3 A Aminoacyl (charged) tRNA P E Ribosomal subunits TRANSLATION E A Anticodon Codon Ribosome Concept 17.5: Mutations of one or a few nucleotides can affect protein structure and function • Mutations are changes in the genetic material of a cell or virus • Point mutations are chemical changes in just one base pair of a gene • The change of a single nucleotide in a DNA template strand can lead to the production of an abnormal protein © 2011 Pearson Education, Inc. Figure 17.23 Wild-type hemoglobin Sickle-cell hemoglobin Wild-type hemoglobin DNA C T T 3 5 G A A 5 3 Mutant hemoglobin DNA C A T 3 G T A 5 mRNA 5 5 3 mRNA G A A Normal hemoglobin Glu 3 5 G U A Sickle-cell hemoglobin Val 3 Types of Small-Scale Mutations • Point mutations within a gene can be divided into two general categories – Nucleotide-pair substitutions – One or more nucleotide-pair insertions or deletions © 2011 Pearson Education, Inc. Substitutions • A nucleotide-pair substitution replaces one nucleotide and its partner with another pair of nucleotides • Silent mutations have no effect on the amino acid produced by a codon because of redundancy in the genetic code • Missense mutations still code for an amino acid, but not the correct amino acid • Nonsense mutations change an amino acid codon into a stop codon, nearly always leading to a nonfunctional protein © 2011 Pearson Education, Inc. Insertions and Deletions • Insertions and deletions are additions or losses of nucleotide pairs in a gene • These mutations have a disastrous effect on the resulting protein more often than substitutions do • Insertion or deletion of nucleotides may alter the reading frame, producing a frameshift mutation © 2011 Pearson Education, Inc. Figure 17.24 Wild type DNA template strand 3 T A C T T C A A A C C G A T T 5 5 A T G A A G T T T G G C T A A 3 mRNA5 A U G A A G U U U G G C U A A 3 Protein Met Lys Phe Gly Stop Carboxyl end Amino end (b) Nucleotide-pair insertion or deletion (a) Nucleotide-pair substitution Extra A A instead of G 3 T A C T T C A A A C C A A T T 5 5 A T G A A G T T T G G T T A A 3 3 T A C A T T C A A A C C G A T T 5 5 A T G T A A G T T T G G C T A A 3 Extra U U instead of C 5 A U G A A G U U U G G U U A A 3 Met Lys Phe Gly Stop Silent (no effect on amino acid sequence) 5 A U G U A A G U U U G G C U A A 3 Met Stop Frameshift causing immediate nonsense (1 nucleotide-pair insertion) T instead of C A missing 3 T A C T T C A A A T C G A T T 5 5 A T G A A G T T T A G C T A A 3 3 T A C T T C A A C C G A T T 5T 5 A T G A A G T T G G C T A A 3A A instead of G U missing 5 A U G A A G U U U A G C U A A 3 Met Lys Phe Ser Stop Missense 5 A U G A A G U U G G C U A A Met Lys Leu Ala Frameshift causing extensive missense (1 nucleotide-pair deletion) A instead of T 3 T A C A T C A A A C C G A T T 5 5 A T G T A G T T T G G C T A A 3 U instead of A 5 A U G U A G U U U G G C U A A 3 Met Nonsense Stop T T C missing 3 T A C A A A C C G A T T 5 5 A T G T T T G G C T A A 3 A A G missing A A 5 A U G U U U G G C U A A 3U Met Phe Gly Stop No frameshift, but one amino acid missing (3 nucleotide-pair deletion) 3 Chromosomal Alteration • • • • • Deletion Duplications – hemoglobin, antifreeze Inversions Translocations Transposons – jumping genes (corn) Gene Expression • Gene expression is the act of going from genotype to phenotype • DNA to mRNA to Protein • Genes are regulated by turning on and off transcription The lac operon in E.coli is the method by which E. coli make enzymes that metabolize lactose Regulatory gene – produces the repressor Repressor binds to the operator when lactose is absent No Transcription – RNA polymerase cannot bind to the promoter When lactose is present, it binds to the repressor pulling it off the operator RNA polymerase binds the promoter – transcription begins Lactose-digesting enzymes are made Regulatory gene DNA Promoter Operator lacI lacZ No RNA made 3 mRNA RNA polymerase 5 Active repressor Protein (a) Lactose absent, repressor active, operon off lac operon DNA lacI lacZ lacY lacA RNA polymerase 3 mRNA 5 mRNA 5 -Galactosidase Protein Allolactose (inducer) Inactive repressor (b) Lactose present, repressor inactive, operon on Permease Transacetylase Trp operon. E.coli will make tryptophan from scratch, but if it is in the surroundings, the E.coli will absorb it. Different from the lac operon trp operon Promoter Promoter Genes of operon DNA trpE trpR trpD trpC trpB trpA C B A Operator Regulatory gene 3 RNA polymerase Start codon Stop codon mRNA 5 mRNA 5 E Protein Inactive repressor D Polypeptide subunits that make up enzymes for tryptophan synthesis (a) Tryptophan absent, repressor inactive, operon on DNA No RNA made mRNA Protein Active repressor Tryptophan (corepressor) (b) Tryptophan present, repressor active, operon off Proximal control elements are located close to the promoter Distal control elements, groupings of which are called enhancers, may be far away from a gene or even located in an intron Some transcription factors function as repressors, inhibiting expression of a particular gene by a variety of methods A particular combination of control elements can activate transcription only when the appropriate activator proteins are present Promoter Activators DNA Enhancer Distal control element Gene TATA box General transcription factors DNAbending protein Group of mediator proteins RNA polymerase II RNA polymerase II Transcription initiation complex RNA synthesis Enhancer Control elements Promoter Albumin gene Crystallin gene LENS CELL NUCLEUS LIVER CELL NUCLEUS Available activators Available activators Albumin gene not expressed Albumin gene expressed Crystallin gene not expressed (a) Liver cell Crystallin gene expressed (b) Lens cell Types of Genes Associated with Cancer • Cancer can be caused by mutations to genes that regulate cell growth and division • Tumor viruses can cause cancer in animals including humans © 2011 Pearson Education, Inc. Cancer Genes • Oncogenes are cancer-causing genes • Proto-oncogenes are the corresponding normal cellular genes that are responsible for normal cell growth and division • Conversion of a proto-oncogene to an oncogene can lead to abnormal stimulation of the cell cycle Evidence That DNA Can Transform Bacteria • The discovery of the genetic role of DNA began with research by Frederick Griffith in 1928 • Griffith worked with two strains of a bacterium, one pathogenic and one harmless © 2011 Pearson Education, Inc. • When he mixed heat-killed remains of the pathogenic strain with living cells of the harmless strain, some living cells became pathogenic • He called this phenomenon transformation, now defined as a change in genotype and phenotype due to assimilation of foreign DNA © 2011 Pearson Education, Inc. Figure 16.2 EXPERIMENT Living S cells (control) Living R cells (control) Heat-killed S cells (control) Mixture of heat-killed S cells and living R cells Mouse healthy Mouse dies RESULTS Mouse dies Mouse healthy Living S cells Animation: Hershey-Chase Experiment Right-click slide / select “Play” © 2011 Pearson Education, Inc. Figure 16.4-3 EXPERIMENT Phage Radioactive protein Empty protein shell Radioactivity (phage protein) in liquid Bacterial cell Batch 1: Radioactive sulfur (35S) DNA Phage DNA Centrifuge Pellet (bacterial cells and contents) Radioactive DNA Batch 2: Radioactive phosphorus (32P) Centrifuge Radioactivity Pellet (phage DNA) in pellet