Genetics EXAM 2 PDF

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Chapter 7: DNA structure and Replication Genetics material must have three key properties: 1. Information content (encoding proteins and RNA) 2. Faithful replication 3. Infrequent change (mutation) What was known when Watson & Crick began their collaboration? • Hereditary factors (genes were associated with specific traits, but their physical nature was not known • Genes specifically the structure of proteins (the gene/one protein hypothesis) • Genes are carries on chromos • Chromosomes consist of DNA and protein • DNA is the genetic material Part 1: DNA is the genetic material • The Griffith Experiment o Experiment with Streptococcus Pneumoniae o Somehow heat killed S strain transform R strain into virulent S strain • The Avery, MacLeod and McCarty experiment o Showed the transforming principle is DNA. Not polysaccharide o The experiment: § Kill S cells à destroy different classes of molecules àadd to R cell o Their findings: § DNA is the transforming agent and is thus the carrier of genetic information • The Hershey-Chase experiment o Final evidence that DNA is genetic material o The T2 phase injects DNA, not protein o (35S)à Radioactive sulfuràall proteins are radioactive o (32P)à Radioactive phosphorusà all DNA are radioactive Part 2: The structure of DNA • How can a molecule with so few components store such a best array of information • An answer to this question, as well as the questions of how genetic information is encoded and replicated, was suggested when James and Watson and Francis Crick solved the structure of DNA in 1953 • It is interesting to note that Watson and Crick never did a single experiment Watson & Crick based their model on the following information: • DNA is composed the four nucleotides: dAMP, dGMP, dCMP, dTMP • Chargaff’s rules (empirical) o Total pyrimidines (T+C)-Total purines (A+G) o A=T and G=C • X-ray diffraction data stolen from Maurice Wilkens and Rosalind Franklin Suggested that: o DNA is a long and skinny molecule that has two similar parts which are parallel to one another along their length (long axis) o The molecule is helical (spiral-like) o The diameter of the molecule is consistent with the pyrimidine+purine at its center, not purine+purine, or pyrimidine+pyrimidine

Watson & Crick came up with a structure that was consistent with the X-ray diffraction data and with Chargaff’s rules • The structure is a double helix: two strands twisted around one another like a spiral staircase • Each strand is a chain of nucleotides (dNMPs) held together by a sugar-phosphate backbone. How the double helix is put together • Each unit in the backbone is linked by 5’ to 3’ phosphodiester bonds. o Phosphodiester means a phosphate forms a bridge between oxygen on two adjacent sugar residues. o 5’ to 3’ denote which carbon atoms are linked on adjacent sugar residues • The two strands (sugar-phosphate backbones) are held together by hydrogen bonds between the two bases o Hydrogen bonds are quite weak, only about 3% the strength of a covalent bond DNA strands are polarà they are assembled in a directional manner (5’ to 3’) Part 3: The Replication of DNA • Phenomenolody (what happens) • Mechanism (how it happens) Phenomenology I: Watson & Crick proposed a semiconservative model of DNA replication but a priori, there are two other possibilities: • Conservative replication • Dispersive replication In 1958, Matthew Meselson and Franklin Stahl proved the semiconservative model of DNA replication in e. coli

Phenomenology II: The Meselson-Stahl Experiment

The Mechanism of DNA Replication DNA is synthesized by DNA polymerase III. Synthesis proceeds only in the 5’ to 3’ direction and nucleotides can only be added to the 3’ end of an existing strand

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The directional nature and primer dependence of DNA synthesis present two problems 1. The leading strand can be synthesized continuously 2. The lagging strand must be synthesized discontinuously

! DNA is replicated in vivo by a large complex of proteins called the “replisome” Steps for lagging (non-template) strand replication: Okazaki fragments 1. Helicase unwinds the strand (breaks H bonds) melting)) 2. Single-strand binding proteins prevent reformation of the duplex. 3. Topoisomerases relax supercoiling 4. RNA primer added by Primase. 5. DNA Polymerase III replicates. A pair of Pol III enzymes catalyze continuous and discontinuous strand synthesis 6. Pol I fills the gaps and degrades the primers. DNA Polymerase I uses its 5’to3’ exonuclease activity to degrade RNA primer and replaces it with DNA 7. Ligase seals the nick by creating the phosphodiester bond (uses ATP) In eukaryotes, chromosomal DNA is highly compacted (by nucleosomes). If stretched out, the DNA in a single human cell would be about 1 meter long. DNA needs to be unwinded to be replicated

Problem Solved: primase and Pol I to the rescue • Chain elongation is catalyzed by DNA polymerase • Pol III is responsible for most of the synthesis • Pol III can only add nucleotides to the 3’ end of existing strands • Primase synthesizes RNA primers that hybridize to sequences along the lagging strand (and the beginning of the leading strand) • Ligase repairs the “nick” • Discontinuous synthesis produces Okazaki fragments

Replication of the E. Coli chromosome is initiated at unique sequences called origins of replication and proceeds in both directions. Eukaryotic cells also employ fixed origins of replication Continuous synthesis: occurs when the polymerase can go across the template in the 3’ to 5’ direction Discontinuous synthesis: template is the wrong way so it has to wait for it to unwind and replicate back the other way

! At the end of the strand, there is a primer left from the lagging strand and it gets degraded leaving the 3’ overhand. But its okay, since we have repeated multiple of sequences at the end of each strand (AAATTTGGGCCC) that do not code of anything and serves as a buffer called telomeres. Telomere become too short after several divisions, they are added by Telomerase Chapter 8: Transcription and Processing

In Eukaryotes: Information transfer: starts with DNA. When DNA is replicated and passed to daughter cells Evidence that RNA serves as the messenger that links DNA to protein. Soon after the discovery of the structure or DNA, it was inferred that there must be at least one information transfer step between DNA and protein because in eukaryotes, DNA is in the nucleus whereas protein was known to be synthesized I the cytoplasm. The two lines of evidence: 1. Pulse-chase experiment 2. The T2 experiments performed in 1957 by Volkin and Astrachan

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First line of evidence: Pulse-chase experiment using 3H-uricil Pulse: tridiated H in Uracil is radioactive, introduced into the medium and cell is growing. All the RNA made is marked with radioactive H- labeled. Chase: Medium is flooded with the cold uracil (non-radioactive). All RNA made after that is unlabeled Result: label appears first in the nucleus then in the cytoplasm, so RNA is a good candidate to be a messenger molecule Conclusion: RNA moves from the nucleus to the cytoplasm and is therefor a good candidate to be the messenger (connecting RNA to protein) Properties of RNA: RNA vs. DNA Two chemical differencesà Ribose instead of Deoxyribose and Uracil instead of Thymine DNA is usually double stranded and RNA is usually single stranded

Transcription: RNA is synthesized by RNA polymerase in 5’ to 3’ direction (adding to 3’ OH end). But RNA polymerase moves on a parent strand in the 3’ to 5’ direction. Transcription is broken down into 4 stages: • Initiation

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Elongation Termination Processing (only in eukaryotes)

1. Initiation: Transcription in prokaryotes: Initiation Consensus sequences: TATA box and CAT box Differences Eukaryotes (more complicated) and prokaryotes transcription: The RNA polymerases that are used to synthesize RNA • The initiation mechanisms • The termination mechanisms • Processing: only eukaryotes do it o The addition of caps and tails o Splicing o Transport Eukaryotes use three distinct RNA polymerases whereas prokaryotes use only one • RNA polymerase I transcribes all the rRNA genes except the 5S. • RNA polymerase II transcribes all mRNAsand some snRNAs. • RNA polymerase III transcribes all tRNAs, some snRNAs, and 5S rRNA. • Eukaryotic RNA polymerases also have a more complex subunit structure and utilize a greater number of cofactors.

Initiation of transcription in Prokaryotes • Sigma-factor “scans” the chromosome. When it encounters the promoter sequence, it helps to melt (unwind) the DNA so that RNA polymerase can start (initiate) transcription. Thus, sigma-factor provides specificity

Initiation of transcription in Eukaryotes • Promoter sequence” TATA-box at -30 • Promoter-recognizing factor: TBP (TATA-Binding Protein) • Several other GTFs (General Transcription Factors) bind to the promoter and attract ad position RNA polymerase II

2. Elongation: the primary difference is In Prokaryotesà translation can begin as soon as the nascent RNA is long enough for ribosomes to bind In Eukaryotes à processing and transport of the RNA must occur before translation In other words, transcription and translation are compartmentalized in Eukaryotes 3. Termination: • Signal: AAUAAA • Factor: endonuclease cuts 20 nucleotides downstream of the signal, and the polymerase falls off the template As compared to prokaryotes in which termination is mediated by a GC-rich hairpin loop and U-rich sequence (intrinsic), or by the rho protein (extrinsic)

4. Processing (Eukaryotes ONLY) Eukaryotic cells process primary RNA transcripts into mature messenger RNAs. Processing occurs in the nucleus in four steps 1. Addition to the 5’ cap 2. Cleavage and termination of transcription 3. Addition of the 3’ poly-A tail 4. Splicing

Processing eukaryotic transcripts: addition of the 5’ cap • Capping the 5’-end of the mRNA has two funtions 1. Prevents degradation of the message 2. Acts as a signal to initiate translation -In many eukaryotic exons, the coding regions (called exon) are interrupted by noncoding regions (called intervening sequences or introns_ - When genes are split in this way, the intervening sequences are actually spliced out as the primary transcript is processed to the mature mRNA - Splicing is catalyzed by the spliceosome, a complex of proteins and snRNAs. à Eukaryotic genes are split • Evidence for introns: Hybridization of RNA with DNA for a specific gene à Electron microscopy • Result: loops of single-stranded DNA Introns are very common in the genes of higher eukaryotes • In the human genome, the average gene contains 7- 8 introns; the average intron size is 2000 bp. • Many genes are composed mostly of introns. o An extreme example is the Duchenne muscular dystrophy gene which has 78 introns totaling 2.5 million base pairs o In contrast, the coding sequence has14,000 bases. o Therefore, only 0.5% of this gene codes for protein! Why do introns exist? Known function of introns: • Introns contain regulatory regions for transcription enhancers and inhibitors. • Introns can contain other genes. • Exon shuffling (important for evolution) • Alternative splicing Because of alternative splicing, one gene can give rise to many different mRNAs, and thus to many different proteins. A given gene might be spliced to yield distinct mRNAs in different cell types and at different stages of development

Self-splicing (autocatalysis) • In 1981 Tom Cech discovered that an rRNA from Tetrahymena splices itself — no protein! • The result was doubted for a long time. Many people insisted there must be some protein contaminant in Cech’s RNA preparations. • Eventually, Cech was proven correct, other examples were found, and he was awarded the Nobel Prize. For a given geneà only one strand of DNA is the template, but different genes on the same chromosome can be on either strand.

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Chapter 9: Protein structure, genetic code, translation

Protein Structure: Proteins are polymers (poly= many; mer=part) made of amino acids attached end to end in a linear string. The general formula for an amino acid is: H2N CHR COOH in which the slide chain, R, can be anything from a hydrogen atom to a complez ring structure.

There are 20 common amino acids found in terrestrial life forms, ech with unique chemical properties that are determined by the R groups. Amino acids are connected by peptide bonds between the carboxyl end of one and the amino end of the next, thus forming a linear polypeptide chain. Proteins have four levels of structure • Primary (1˚) structure refers to the linear sequence of amino acids in a polypeptide o the sequence of amino acids in a chain • Secondary (2˚) structure refers to interactions between amino acids that are close together. 2˚ structure is usually due to hydrogen bonding between the CO and NH groups of nearby amino acid residues. Examples include ahelices and b-pleated sheets. o The first level of 3D folding; determined by H-bonding between nearby NH and CO groups o The two most common types of secondary structure: a-helix and b-pleated sheet • Tertiary (3˚) structure refers to the higher order folding of a protein. It is determined by electrostatic, hydrogen, and Van der Waals bonds between R groups (often distant). o The folding of the secondary structures to form the final 3D shape of a polypeptide • Quaternary (4˚) structure refers to the binding together of two or more individual polypeptides to form a multimeric protein (e.g., hemoglobin). o Several polypeptides join together to form a multi-subunit structure o Hemoglobin is made up of two a and two b chains Protein structure summary • Primary structure (the sequence of amino acids) determines the secondary and tertiary structures of the protein (the way the string of amino acids folds in 3D space). • The amino acid side chains determine the folding of the protein, and provide functionality to the interaction surfaces and to the active sites of enzymes. The genetic code: Chapter 11: Chapter 12:

Chapter 10: Cutting of DNA molecules • Molecular biolist cut DNA with Restriction enzymes o REs cut strands of DNA by breaking the covalent (phoshophodiester bonds) between the deoxyribose units that form the backbone • How is DNA metabolized o Examples of nucleic acid metabolism: replication, homologous recombination (crossing over), DNA modification, packaging, RNA processing (capping, splicing, polyadenylation, degradation) o Some of the enzymes that do the work: polymerases, helicases, ligases, exonucleases, endonucleases (REs), methylases, transferases o These naturally occurring enzymes are the foundation of RDT. • Res binds to specific DNA sequences (recognition sites) and cut between specific nucleotides (cut sites), thus generating characteristic ends o Some Res generate “sticky” ends: others yield “blunt” (even) ends o Recognition site is not the same as cut site! Restriction enzymes (DNA endonucleases) • EcoRI generates sticky (overhang) ends. • Both circular (plasmid) and linear DNA molecules can be cut. • Some REs recognize 6 bp sequences (six cutters); others recognize 4 bp sequences (four cutters). • Remember, the recognition site ≠ cut site • In theory, how frequently would you expect the recognition site for a given four cutter to occur in a random sequence? A six cutter? • Why are such predictions not always very accurate? • Sticky ends are sticky because they are single strands with complementary base pairs. Blunt ends have no such single-stranded overhang The sequences recognized by most restrictiong enzymes are palindromic • Definition of a palindrome, An analogy … • Example of a DNA palindrome, the EcoRI recognition site: 5'GAATTC-3' 3'-CTTAAG-5' • A limitation in the analogy • Palindromic sequences have 2-fold rotational symmetry. Why do you suppose REs recognize such sequences? • Hundreds of REs have been purified. JOINING of DNA molecules • The joining of two DNA molecules by covalent linkage of their deoxyribose backbones is catalyzed by an enzyme called DNA ligase. • DNA ligase can join DNA molecules that have blunt ends as well as those that have sticky ends, as long as there is a 5' phosphate and a 3' OH in the gap. Insertion of donor DNA into a plasmid a. The donor and plasmid DNA are cut with compatible REs b. The sticky ends “hybridize” by virtue of complementary base-pairing, but there is a “gap” in the backbone (there is no phosphodiester bond) c. Ligase seals the gap in the backbone REPLICATION and AMPLIFICATION of DNA • Recombinant plasmid DNA is introduced into host cells by transformation (see chapter 5 and below), where it is replicated by cellular enzymes and factors (see chapter 7). Transformed cells are selected by a plasmid-borne marker. • Amplification occurs by two mechanisms: – All cells in the culture contain at least one copy of the plasmid. Typically, E. coli grows to 109 cells/ml. – Many plasmids are maintained in multiple copies per cell. • Only circular DNA molecules can be replicated autonomously. Linear DNA molecules must integrate into the host cell’s chromosome, or they are lost.

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How to isolate a specific DNA sequence, two approaches 1. The library method: DNA fragments carried on plasmid or in phage are amplified by E. coli and screened for the desired sequence 2. PCR amplification of a specific sequence: DNA polymerase is used with specific primers to synthesize a specific fragment Confusion over the work CONING Meaning 1: production of genetically identical organisms from somatic cells (dolly) Meaning 2: the isolation and amplification of a particular DNA sequence: - Finding a sequence corresponding to a specific gene - Amplifying this gene by inserting it into a plasmid Cloning a specific gene usingthe library method • The goals of recombinant DNA technology reprised – o Isolation and amplification of specific DNA segments from large complex samples – o Recombination of various DNA segments for experimental purposes and for application in medicine, forensics, industry, and agriculture • • Genome sizes – o E. coli: 4.7 X 106 base pairs o S. cerevisiae: 1.3 X 107 base pairs o D. melanogaster: 6.0 X 107 base pairs o C. elegans: 6.0 X 107 base pairs o H. sapiens: 3.0 X 109 base pairs per one set of chromosomes. • To isolate and purify a specific gene, you must do two things: o Break up the genome into small (gene-sized) pieces. In other words, you have to create a library in a cloning vector. o Identify the piece of interest. For this, you need a detection method. What are cloning vectors? • Cloning vectors carry foreign pieces of DNA, which allows them to be replicated and amplified in cellular systems. • In order to be useful, cloning vectors must . . . • be greatly amplified by replication when introduced into cells – have convenient restriction sites • provide a means of identifying recombinant molecules. • There are four basic types of cloning vectors: plasmids, l phage, cosmids (fosmids), and artificial chromosomes. Types of cloning vectors • Plasmid vectors o The most convenient, small size (inserts up to 11 kb*), easy manipulation *kb = kilobase • Bacteriophage vectors o Allow larger inserts, easy to infect into bacteria, high level of protein production Plasmids o Plasmids are small (up to 15 kb) circular pieces of DNA constructed from naturally occurring molecules (e.g., the F factor discussed in chapter 5). o All plasmids have: o An origin of replication (which determines copy number) o A polylinker/MCS (multiple cloning site with unique cut sites) o At least one selectable marker (because transformation is inefficient) o A means to detect inserts (because ligation is inefficient) such as: § Blue/white screening (Fig. 10-9) § Disruption of a selectable marker...