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BL 426 Molecular Biology

BL 426 Molecular Biology

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BL 426 Molecular Biology

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  1. BL 426 Molecular Biology • What is molecular biology? • explain biological phenomena in molecular terms • study gene structure, function at molecular level • Melding of genetics, microbiology and biochemistry • Dates from about 1940s to 1960s • Techniques permitted incredible details of basic science; many practical applications in medicine, agriculture

  2. Learning Outcomes for Students: • Generally explain how science differs from other ways of knowing – experimental basis for conclusions • Define major terms used in Molecular Biology • Explain major organizing concepts in Molecular biology. • Recognize social and ethical relevance of content covered in Molecular biology • Analyze and present primary scientific data, research by Nobel Laureates in Medicine or Chemistry.

  3. Chapt. 1 Brief History 1.1 Transmission Genetics (Mendelian): • Transmission of traits from parents to offspring • Chemical composition of genes discovered 1944; Mendel didn’t know chromosomes • Gene – particles contributed by parents • Phenotype – observed characteristics Mendel studied garden pea, detailed records, statistics Important Figure 3, Table 1

  4. Mendel’s Laws of Inheritance • Genes exist in different forms - alleles • One allele (A) dominantover other, recessive (a) • Each parent carries 2 copies of gene: diploid for that gene: • Parents in 1st mating are homozygotes (AA, aa) • First filial generation (F1) contains offspring of parents • Heterozygotes have one copy of each allele: (Aa) • Sex cells, or gametes, are haploid, contain 1 copy of gene • Heterozygotes produce gametes having either allele • Homozygotes produce gametes having only one allele • Recall Punnet square analysis to predict progeny

  5. Chromosome Theory of Inheritance • Chromosomes: discrete physical entities that carry genes • Morgan used fruit fly, Drosophila melanogaster, to study genetics • Autosomes occur in pairs in individual • Sex chromosomesare X and Y • Female has two X chromosomes • Male has one X and one Y

  6. Hypothetical Chromosomes • Every gene has its place, or locus, on chromosome centromere attaches to spindle • Genotype: combination of alleles found in organism • Phenotype: visible expression of genotype • Wild-type phenotype - most common, generally accepted standard • Mutant alleles – altered, usually recessive Fig. 3

  7. Genetic Recombination and Mapping • Genes on separate chromosomes behave independently • Genes on same chromosome behave as if linked • Genetic linkage is not absolute: permits mapping • Recombination produces new combinations of alleles in offspring, combinations not seen in parents • from Crossing-overof chromosomes during meiosis • Genetic Mapping: farther apart two genes are on chromosome, more likely they are to recombine (Fig. 4) • If 2 loci recombine with frequency of 1%: map distance is 1 centimorgan (named for Morgan) • (mapping applies to Prokaryotes and Eukaryotes)

  8. 1.2 Molecular Genetics overview • Discovery of DNA: general structure of nucleic acids found by end of 19th century Long polymers or chains of nucleotides Nucleotides linked by sugars through phosphate groups • Composition of Genes: In 1944, genes are composed of nucleic acids Genes perform three major roles: • Replicate faithfully • Direct production of RNAs and proteins • Accumulate mutations, thereby allowing evolution

  9. DNA Replication • Franklin and Wilkins x-ray diffraction data on DNA • Watson and Crick proposed DNA is double helix • Two DNA strands wound around each other • Strands are complementary – • if know sequence of one, automatically know sequence of other • Semiconservative replication: one strand of parental double helix conserved in each daughter double helix

  10. Genes Direct Production of Polypeptides • Defective gene gives defective or absent enzyme • Early idea: one gene makes one enzyme • Gene expression - process making gene product: • Transcription: copy of DNA is made as RNA • Translation: RNA copy is read or translated to assemble a protein (on ribosomes) • Codon: sequence of 3 nucleic acid bases that stand for 1 amino acid

  11. Genes Accumulate Mutations Genes change in several ways: • Change one base to another • Deletions of one base up to a large segment • Insertions of one base up to a large segment • Rearrangements of chromosomes • The more drastic changes make it more likely that gene or genes involved will be totally inactivated

  12. 1.3 Three Domains of Life Current research supports division of living organisms into three domains • Bacteria – typical prokaryotes: E. coli; Thermusaquaticus • Eukarya – nucleus, organelles: yeast, amoeba, worms, mice, humans • Archaea(prokaryotes) often live in inhospitable regions of earth • Thermophiles tolerate extremely high temperatures Thermococcus • Halophiles tolerate very high salt concentrations Halobacterium

  13. Chapt. 2 DNA is Genetic Material Learning outcomes: • Recall and explain basic experiments, concepts of DNA as basis of heredity; • Describe general structure of DNA and RNA: Important Figures: 2, 4, 5, 6, 7, 8, 9, 10*, 11*, 13, 14*, 15, 20 Table 4 Review Q 2, 3, 4, 8; AQ 1, 2 • chain-like molecules composed of nucleotide subunits • Nucleotides contain a base linked to the1’-position of a sugar and a phosphate group • Phosphate joins sugars in DNA or RNA chain through 5’- and 3’-hydroxyl groups by phosphodiester bonds • (be able to draw Fig. 10 details)

  14. DNA is Genetic Material Bacterial Transformation – Griffith, 1928; Avery, 1944 Fig. 2

  15. DNA Confirmation • In 1940s geneticists doubted importance of DNA: appeared monotonous repeats of 4 bases • 1950 Chargaff showed 4 bases were not present in equal proportions • 1952 Hershey and Chase demonstrated (S35, P32) that bacteriophage T2 infection comes from DNA • 1953 Watson & Crick published double-helical model of DNA structure • Genes are made of nucleic acid, usually DNA • Some simple genetic systems (viruses) have RNA genes

  16. DNA is phage T2 genetic materialHershey – Chase 1952 Fig. 4 Phage T2

  17. Purines and Pyrimidines • A and G are purines; C, T and U are pyrimidines • Note numbering of positions Fig. 5

  18. Nucleosides and Nucleotides • RNA component parts • Nitrogenous bases • Uracil (U) • replaces Thymine • Phosphoric acid • Ribose sugar • Bases had ordinary numbers • Carbons in sugars - primed numbers • Nucleosides lack phosphate • Nucleotides contain phosphate Fig. 7

  19. DNA nucleotide linkage • Nucleotides are nucleosides with phosphate group attached through phosphodiester bond • Nucleotides may contain 1, 2, or 3 phosphate groups Fig. 9

  20. Trinucleotide: phosphodiester bond Polarity: 5’- T-C-A-3’ • Top of molecule has free 5’-phosphate group = 5’ end • Bottom has free 3’-hydroxyl group = 3’ end Figs. 10, 11

  21. DNA Double Helix • Twisted ladder structure: • Curving sides of ladder are sugar-phosphate backbone • Ladder rungs are base pairs • A-T and G-C hydrogen bond • About 10 base pairs per turn • Two strands are antiparallel Fig. 13 Fig. 14

  22. Genes can be made of RNA or DNA Hershey & Chase investigated bacteriophage T2 (virus particle, DNA,package of genes) • T2 has no metabolic activity of its own • When virus infects host, cell makes viral proteins • Viral genes are replicated, newly made genes with viral coat proteins assemble into virus particles Viruses are model systems for molecular biology: • Some viruses contain DNA genes, either single- or double-stranded (M13, lambda) • Some viruses have RNA genes, either single- or double-stranded (MS2, HIV, rabies)

  23. DNA Content varies among Organisms • Ratios of G to C, A to T are fixed in any organism • But, total percentage of G + C varies over a range of 22 to 73% • Differences in total G+C reflected in differences in physical properties (such as melting temp)

  24. Polynucleotide Chain Hybridization ** Hybridization: process of putting together combination of two different nucleic acids • Strands could be 1 DNA and 1 RNA • Could be 2 DNAs • Could be complementary or nearly complementary sequences • Valuable technique Fig. 20

  25. DNA Shapes and Sizes DNA size is expressed 3 different ways: • Number of base pairs (bp, kbp or kb) • Molecular weight – 660 daltons (D) is average molecular weight of 1 base pair • Length – 33.2 Å per helical turn of 10.4 base pairs DNA shape can be linear, circular (relaxed), or covalently closed circular Measure DNA size (shape) using electron microscopy or gel electrophoresis

  26. Phage DNA is typically circular; so are bacterial chromosomes • Some DNAs are linear – ex., eukaryotic chromosomes • Supercoiled DNA coils or wraps around itself like a twisted rubber band

  27. Ex. DNA Size and Genetic Capacity Estimate how many genes are in a piece of prokaryotic DNA • Gene encodes protein; avg. Protein is about 40,000 D (40 kD) • How many amino acids does this represent? • Average mass of an amino acid is about 110 D • Average protein of 40,000 / 110 = 364 amino acids • Each amino acid = 3 DNA base pairs • 364 amino acids requires 1092 base pairs E. coli chromosome = 4.6 x 106bp; ~4200 proteins • Phage l (infects E. coli) = 4.85 x 104bp~44 proteins • Phage x174 (one of smallest) = 5375 bp~5 proteins

  28. Chapt. 3 Gene Function Learning outcomes: • Recall and explain basic processes in production of polypeptide from DNA • transcription • translation • ribosome • tRNA • mRNA • polypeptide, protein structure (1o, 2o, 3o, 4o) Important Figures: 1, 2a, 3, 4, 14, 16, 17, 18, 19, 20*, 26 Review Q: 1, 2, 4, 7, 9, 11, 12, 13*, 14*, 15*; AQ 1

  29. Chapt. 3 Gene Function 3.1 Storing Information Producing protein from DNA involves both transcription and translation • A codon is 3 base sequence that determines what amino acid • Template strand: complementary DNA strand used to generate mRNA • Nontemplatestrand: not used for RNA but = sequence of mRNA (with U for T) Fig. 1

  30. Polypeptides (proteins) • Amino acids joined together with peptide bonds • Chains of amino acids are polypeptides • Proteins are composed of 1 or more polypeptides • Polypeptides have polarity (as does DNA) • Free amino group at one end is amino- (N-terminus) • Free acid group at other end is carboxyl- (C-terminus) Fig. 3

  31. Protein Structure • Proteins: polymers of amino acids linked through peptide bonds • Sequence of amino acids (primary structure) gives rise to molecule’s: • Local shape (secondary structure) • Common types of secondary structure: H bonds of nearby backbone • a-helix • b-sheet • Overall shape (tertiary structure) • Interaction with other polypeptides (quaternary structure)

  32. Secondary Structures - Tertiary structure Figs. 4, 5 Fig. 6; myoglobin helix, pleated sheet Interaction of aa side chains – longer range

  33. Protein Domains • Compact structural regions of protein are domains • Immunoglobulins example of 4 globular domains (Fig. 8) • Domains may contain common structural-functional motifs: • Zinc finger • Hydrophobic pocket • Quaternary structure is interaction of 2 or more polypeptides

  34. Relationship Between Genes and Proteins one gene - one polypeptide hypothesis: Most genes contain information for making 1 polypeptide • 1902 suggestion link between disease alkaptonuria and recessive gene • If a single gene controlled production of an enzyme, lack of enzyme could result in buildup of homogentisic acid, which is excreted in urine • If gene responsible for enzyme is defective, then enzyme ly also is defective • Many enzymes contain more than one polypeptide chain: • Each polypeptide is usually encoded in one gene

  35. mRNA is Information Carrier • mRNAs carry genetic information from genes to ribosomes, which synthesize polypeptides • In 1950s and 1960s, concept of messenger RNA -carries information from gene to ribosome: • Intermediate carrier needed: in eukaryotes, DNA in nucleus, proteins made in cytoplasm • Jacob & Monod, from genetic experiments, proposed ribosomes translate unstable RNAs called messengers; • Messengers are independent RNAs that move information from genes to ribosomes

  36. Transcription • Transcription follows same base-pairing rules as DNA replication • U replaces T in RNA • Base-pairing pattern ensures RNA transcript is faithful copy of gene • For transcription to occur at a significant rate, reaction is enzyme-mediated • RNApolymerase (RNAP) is enzyme directing transcription

  37. Synthesis of RNA Fig. 20; RNAP uses bp rules

  38. Transcription Phases Initiation: RNAP binds, local melting, First few phosphodiester (asymmetric synthesis) Elongation: RNAP links more ribonucleotides 5’-> 3’ Termination: RNAP, RNA and DNA template dissociate Fig. 14; much more detail later

  39. Transcription Landmarks • RNA sequences written 5’ to 3’, left to right • Translation occurs 5’ to 3’; ribosomes reading mRNA 5’ to 3’ • Genes written so that transcription proceeds from left to right • Gene’s promoter area lies just before start site, said to be upstream of transcription • Genes lie downstream of promoters 5’ _____P_____+1____ORF_____________ -3’ up down

  40. Translation on Ribosomes • Ribosomes are cell’s protein factories • Bacteria contain 70S ribosomes (Euks 80S) • Each ribosome has 2 subunits • 50 S • 30 S • Each subunit contains rRNA and many proteins • No translation of rRNAs Fig. 16

  41. Transfer RNA: Adapter Molecule • tRNA: small RNA recognizes both mRNA and amino acids • Cloverleaf model of tRNA structure/function: • One end (top, 3’ end) binds specific amino acid • Bottom end contains 3 bp sequence (anticodon)that pairs with complementary sequence of mRNA (codon) Fig. 17

  42. Codons and Anticodons • Enzymes that catalyze attachment of amino acid to tRNA are aminoacyl-tRNAsynthetases • A triplet in mRNA is codon • Complementary sequence to codon found in tRNA is anticodon Fig. 18

  43. Initiation of Protein Synthesis • Initiation codon (AUG) interacts with special aminoacyl-tRNA • In eukaryotes methionyl-tRNA • In bacteria N-formylmethionyl-tRNA • Position of AUG codon: • At start of message AUG is initiator • In middle of message AUG is regular methionine • In Bacteria, Shine-Dalgarno sequence lies just upstream of the AUG, functions to attract ribosomes • Eukaryotes have special cap on 5’-end of mRNA; ribosomes bind and find AUG

  44. Translation Elongation • Initiatingaminoacyl-tRNA binds to P site on ribosome • Amino acids added one at a time to initiating amino acid • First elongation step is binding of second aminoacyl-tRNA to A site on ribosome: • Process requires: • elongation factor, EF-Tu • Energy from GTP Fig. 19

  45. Termination of Translation; mRNA Structure • 3 different codons (UAG, UAA, UGA) cause translation termination • Protein release factors recognize stop codons, cause: • Translation to stop • Release of polypeptide chain • Initiation codon and termination codon at ends define an open reading frame (ORF)

  46. **Structural Relationship Between Gene, mRNA and Protein Transcription of DNA does not begin or end at same places as translation • Ex. Transcription • begins at first G • Translation begins 9-bp downstream • This mRNA has 9-bp leader or 5’-untranslated region 5’-UTR Fig. 20

  47. Structural Relationship Between Gene, mRNA and Protein, cont. Trailer sequence is present at 3’ end of mRNA • between stop codon and transcription termination site • This mRNA has a 3’-untranslated region or a 3’-UTR

  48. 3.3 Mutations • Genes accumulate changes or mutations • Mutation is essential for evolution • If a nucleotide in a gene changes, likely a corresponding change will occur in an amino acid of that gene’s protein product • If a mutation results in a different codonfor the same amino acid it is a silent mutation • Often a new amino acid is structurally similar to the old and the change is conservative