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BL 426 Molecular Biology What is 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) dominant over 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 chromosomes are 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-over of 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; Thermus aquaticus 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



15 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 the 1’-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)

16 Bacterial Transformation – Griffith, 1928; Avery, 1944
DNA is Genetic Material Bacterial Transformation – Griffith, 1928; Avery, 1944 Key experiments by Griffith in 1928: Observed change in Streptococcus pneumoniae — from virulent (S) smooth colonies where bacteria had capsules, to avirulent (R) rough colonies without capsules Heat-killed virulent cells could transform avirulent cells to virulent ones In 1944 Avery confirmed, extended Griffith’s results, defined chemical nature of the transforming substance: Techniques used excluded both protein and RNA as chemical agent of transformation Other treatments verified that DNA is chemical agent of transformation of S. pneumoniae from avirulent to virulent Fig. 2

17 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

18 DNA is phage T2 genetic material Hershey – Chase 1952
Fig. 4 Phage T2

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

20 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

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

22 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

23 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

24 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)

25 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)

26 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

27 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

28 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

29 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 106 bp; ~4200 proteins Phage l (infects E. coli) = 4.85 x 104 bp ~44 proteins Phage x174 (one of smallest) = 5375 bp ~5 proteins

30 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

31 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 Nontemplate strand: not used for RNA but = sequence of mRNA (with U for T) Fig. 1

32 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

33 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)

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

35 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

36 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

37 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

38 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 RNA polymerase (RNAP) is enzyme directing transcription

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

40 Transcription Phases Initiation: (asymmetric synthesis) Elongation:
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

41 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

42 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

43 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

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

45 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

46 Translation Elongation
Initiating aminoacyl-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

47 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)

48 **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

49 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

50 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 codon for 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

51 Example: Sickle Cell Disease
Sickle cell disease genetic disorder (recessive) Disease results from single base change in gene for b-globin (missense mutation: GAG -> GTG) insertion of incorrect amino acid into position of b-globin protein (Glu -> Val) Altered protein distortion of red blood cells under low-oxygen conditions change in gene causes change in protein product Fig. 25

52 Review questions Chapts. 2 and 3
2.3. Draw structure of phosphodiester bond, with flags for bases, sugars clear, deoxy position indicated. 2.8 Draw principle of nucleic acid hybridization. 2AQ2 How many proteins of average size encoded by phage T2 with 150,000 bp genome (assume no overlap) 3.2 Draw the structure of the peptide bond. 3.7 Describe the two main steps in gene expression. 3.12 How does tRNA serva as adaptor between the 3-bp codons in mRNA and amino acids in proteins? Consider single base mutations, and explain how they could lead to premature termination of mRNA, to change reading frame, or to substitute an amino acid.

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