Ch 10 Molecular Biology of the Gene

Ch 10 Molecular Biology of the Gene For Next Week Lab -Hand in questions from 4 and 5 by TUES in my mailbox (Biology Office) -Do questions for Lab 6 for next week -Lab practical next week Lecture Read chapters 10, 11 1

Until now What is the genetic material? Proteins or DNA? 2

Head DNA Tail Tail fiber Hershey-Chase Experiment Hershey-Chase Experiment http://highered.mcgrawhill.com/sites/dl/free/0072437316/120076/ bio21.swf 3

Phage Bacterium Radioactive protein Empty protein shell Phage DNA Radioactivity in liquid DNA Batch 1 Radioactive protein Centrifuge Pellet 1 Mix radioactively labeled phages with bacteria. The phages infect the bacterial cells. 2 Agitate in a blender to separate phages outside the bacteria from the cells and their contents. 3 Centrifuge the mixture so bacteria form a pellet at the bottom of the test tube. 4 Measure the radioactivity in the pellet and the liquid. Batch 2 Radioactive DNA Radioactive DNA Centrifuge Pellet Radioactivity in pellet The components of DNA and RNA 4

The components of DNA and RNA The monomer unit of DNA and RNA is the nucleotide, containing Nitrogenous base 5-carbon sugar Phosphate group DNA and RNA are polymers called polynucleotides A sugar-phosphate backbone Nitrogenous bases extend from the sugarphosphate backbone Copyright 2009 Pearson Education, Inc. Phosphate group Nitrogenous base Sugar Sugar-phosphate backbone DNA nucleotide Phosphate group Nitrogenous base (A, G, C, or T) Thymine (T) Sugar (deoxyribose) DNA nucleotide DNA polynucleotide 5

Thymine (T) Pyrimidines Cytosine (C) Adenine (A) Purines Guanine (G) Phosphate group Nitrogenous base (A, G, C, or T) Thymine (T) Sugar (deoxyribose) 6

Phosphate group Nitrogenous base (A, G, C, or U) Uracil (U) Sugar (ribose) Uracil Guanine Adenine Cytosine Phosphate Ribose 7

The structure of DNA 8

Watson, Crick and Franklin Franklin: sugar phosphate backbones must be on outside of double helix, forcing nitrogenous bases to swivel in the interior Twist 9

Watson, Crick and Franklin How are bases arranged? If like bases pair with like, DNA molecule would have uniform diameter. NO! Double ringed purine has to pair with single ringed pyridimine! A pairs with T C pairs with G Explained what Chargaff had observed Base pair Hydrogen bond Ribbon model Partial chemical structure Computer model 10

DNA electrophoresis Important for DNA profiling (analyzing DNA fragments to determine if they come from a particular individual) Used to separate DNA fragments based on size First, cut DNA in specific places using restriction enzymes. In DNA profiling, typically use areas of repetitive DNA (between genes) Vary considerably between individuals Run through a gel with an electric current. Because of the negative charge of the phosphate group, the DNA will move towards the positive side of the apparatus. Longer molecules move slowly, shorter molecules move faster. http://www.mtholyoke.edu 11

DNA replication DNA replication DNA replication follows a semiconservative model The two DNA strands separate Each strand is used as a pattern to produce a complementary strand, using specific base pairing Each new DNA helix has one old strand with one new strand Copyright 2009 Pearson Education, Inc. 12

Parental molecule of DNA 13

Nucleotides Parental molecule of DNA Both parental strands serve as templates Parental molecule of DNA Nucleotides Both parental strands serve as templates Two identical daughter molecules of DNA 14

DNA replication Starts at sites called origins of replication where proteins (helicase) attach to DNA and separate strands DNA replication then proceeds in two different directions DNA replication Two strands of DNA run in opposite directions Each strand has a 5 end and a 3 end The 3 end has a free hydroxyl group attached to the 3 carbon of the sugar, while the 5 end has a free phosphate attached to the 5 carbon of the sugar. Important for replication! 15

5 end P 5 4 3 1 2 P 3 end 2 3 1 4 5 P P P P P P 3 end 5 end DNA replication DNA polymerases link DNA nucleotides to the 3 end, so copy can continuously grow in the direction 5 3 DNA replication occurs in the 5 3 direction Replication is continuous on the 3 5 original (leading strand) Replication is discontinuous on the 5 3 original, forming short segments that are joined together by DNA ligase (lagging strand) 16

5 3 Parental DNA DNA polymerase molecule 3 5 3 5 Daughter strand synthesized continuously Daughter strand synthesized in pieces 5 3 DNA ligase Overall direction of replication Origin of replication Parental strand Daughter strand Bubble Two daughter DNA molecules 17

DNA replication Proteins involved in DNA replication DNA polymerase adds nucleotides to a growing chain DNA ligase joins small fragments into a continuous chain Copyright 2009 Pearson Education, Inc. Origin of replication Parental strand Daughter strand Bubble Two daughter DNA molecules 18

5 3 Parental DNA DNA polymerase molecule 3 5 3 5 Daughter strand synthesized continuously Daughter strand synthesized in pieces 5 3 DNA ligase Overall direction of replication The Flow of Genetic Information: The Central Dogma 19

DNA Nucleus Cytoplasm DNA Transcription RNA Nucleus Cytoplasm 20

DNA Transcription RNA Nucleus Cytoplasm Translation Protein DNA strand Transcription RNA Translation Codon Polypeptide Amino acid 21

Second base First base Third base Transcription From DNA - RNA 22

Transcription Overview of transcription The two DNA strands separate One strand is used as a pattern to produce an RNA chain, using specific base pairing For A in DNA, U is placed in RNA RNA polymerase catalyzes the reaction, binding to a promoter region of DNA, continuing to elongate the RNA molecule, and eventually detaching when it reaches the terminator of the DNA template Copyright 2009 Pearson Education, Inc. Transcription Stages of transcription Initiation: RNA polymerase binds to a promoter, where the helix unwinds and transcription starts Elongation: RNA nucleotides are added to the chain Termination: RNA polymerase reaches a terminator sequence and detaches from the template Copyright 2009 Pearson Education, Inc. 23

RNA polymerase RNA nucleotides Direction of transcription Newly made RNA Template strand of DNA RNA polymerase DNA of gene Promoter DNA 1 Initiation Terminator DNA 2 Elongation Area shown in Figure 10.9A 3 Termination Growing RNA Completed RNA RNA polymerase 24

mrna Messenger RNA (mrna) contains codons for protein sequences In Prokaryotes, transcription and translation occur in same place In Eukaryotes, transcription in nucleus, translation in cytoplasm need to prep mrna! Eukaryotes prepping RNA Add a cap and a tail to facilitate exportation, protect from enzymes Eukaryotic mrna has interrupting sequences called introns, separating the coding regions called exons Eukaryotic mrna undergoes processing before leaving the nucleus Addition of cap and tail RNA splicing: removal of introns and joining of exons to produce a continuous coding sequence 25

DNA RNA transcript with cap and tail Cap Exon Intron Exon Intron Exon Transcription Addition of cap and tail Introns removed Tail mrna Exons spliced together Coding sequence Nucleus Cytoplasm Translation 26

Important player in translation: trna Transfer RNA (trna) molecules match an amino acid to its corresponding mrna codon trna structure allows it to convert one language to the other trna Made of single RNA strand (~80 nucleotides), which folds in on itself Single stranded loop at top w/special triplet of bases (anticodon) Other end site where amino acid can attach 27

An amino acid attachment site allows each trna to carry a specific amino acid An anticodon allows the trna to bind to a specific mrna codon, complementary in sequence A pairs with U, G pairs with C trna Another Important Player in Translation: The Ribosome Contains protein and another RNA (called rrna) Large subunit trna-binding sites Specific binding site for mrna Two binding sites for trna mrna binding site Small subunit 28

1. INITIATION Stages of Translation 1. mrna binds to small ribosomal subunit, initiator trna binds to start codon of mrna 2. Large ribosomal subunit binds to small, initiator trna fits into P site (holds growing polypeptide), A site ready for next trna Initiator trna Met Met Large ribosomal subunit P site A site mrna 1 Start codon Small ribosomal subunit 2 1. ELONGATION Stages of Translation 1. Amino acids added one by one 2. Anticodon of trna pairs with mrna in A site 3. Polypeptide separates from trna in P site to new one in A site 4. P site trna leaves, one in A site moves to P site 5. Elongation continues 29

1. TERMINATION Stages of Translation 1. Elongation continues until stop codon is reached in A site 2. Completed polypeptide released, exits ribosome 3. Ribosome splits into subunits again Polypeptide Amino acid mrna P site Codons A site Anticodon 1 Codon recognition mrna movement Stop codon New peptide bond 2 Peptide bond formation 3 Translocation 30

DNA Transcription mrna RNA polymerase 1 mrna is transcribed from a DNA template. Amino acid Translation Enzyme 2 Each amino acid attaches to its proper trna with the help of a specific enzyme and ATP. trna ATP Anticodon Initiator trna mrna Start Codon Large ribosomal subunit Small ribosomal subunit 3 Initiation of polypeptide synthesis The mrna, the first trna, and the ribosomal sub-units come together. Growing polypeptide New peptide bond forming mrna Codons 4 Elongation A succession of trnas add their amino acids to the polypeptide chain as the mrna is moved through the ribosome, one codon at a time. Polypeptide Stop codon 5 Termination The ribosome recognizes a stop codon. The polypeptide is terminated and released. Mutations A mutation is a change in the nucleotide sequence of DNA Base substitutions: replacement of one nucleotide with another Effect depends on whether there is an amino acid change that alters the function of the protein Deletions or insertions Alter the reading frame of the mrna, so that nucleotides are grouped into different codons Lead to significant changes in amino acid sequence downstream of mutation Cause a nonfunctional polypeptide to be produced Copyright 2009 Pearson Education, Inc. 31

Normal hemoglobin DNA Mutant hemoglobin DNA mrna mrna Normal hemoglobin Glu Sickle-cell hemoglobin Val Normal gene mrna Protein Met Lys Phe Gly Ala Base substitution Met Lys Phe Ser Ala Base deletion Missing Met Lys Leu Ala His 32