BY450 Gene Are DNA Fall 2023 PDF

Genes Are DNA and Encode RNAs and Polypeptides (Section I) BY 450 Fall 2023  Chromosomes In eukaryotic species, most of the genetic material is found inside of the nucleus. Instead of one contiguous strand of DNA, the genetic mater...

Genes Are DNA and Encode RNAs and Polypeptides (Section I) BY 450 Fall 2023  Chromosomes In eukaryotic species, most of the genetic material is found inside of the nucleus. Instead of one contiguous strand of DNA, the genetic material is most often divided into units called chromosomes.  Chromosomes In order to organize and compact this genetic information into a size that can fit inside of the nucleus: ❑ DNA is wrapped around “spools” to form organizational units called nucleosomes. Nucleosome = DNA + protein (histone) ❑ Approximately 200 base pair (bp) of DNA can be spooled around a single histone (67 nm DNA per histone). Nucleosome  Chromosomes Nucleosomes are further coiled (and supercoiled) to form a solenoid structure. ❑ Involves linker histones ❑ These resulting solenoids can then be further compacted on a protein scaffold.  DNA: The Genetic Material Mendel’s experiments demonstrated indirect, but observable, evidence for how heritable information (i.e., genes) are transmitted from one generation to the next. Early 20th century, still remained a mystery as to the exact physical basis of inheritance. ❑ What chemical substance encodes the instructions for the heritable traits?  DNA: The Genetic Material A number of pioneering experiments in the early 20th century provided evidence that suggested the nucleic acids were responsible for the transmission of traits For quite some time it was thought that proteins were the most likely candidate for the transmission of traits from one generation to another. ❑ As recent as the first half of the 20th century, there was must resistance to the hypothesis that DNA was the molecule of heredity.  DNA: The Genetic Material Frederick Griffith (1928) ❑ Model organism: Streptococcus pneumoniae Causes pneumonia (humans) Infections are typically lethal in mice ❑ Though normally lethal, he observed that certain colonies were less virulent (sub-lethal effects in mice)  DNA: The Genetic Material Frederick Griffith (1928) ❑ Observed that certain colonies were less virulent (sub-lethal effects in mice) Noticed that there were morphological differences in the strains as well: ❑ Virulent strain = smooth appearance (polysaccharide capsule); designated this as the S strain ❑ Nonvirulent strain = rough appearance (no capsule) designated this as the R strain  DNA: The Genetic Material Frederick Griffith (1928) ❑ Experimental design Step 1 ❑ Injection of virulent (S) strain into mouse = death ❑ Injection of nonvirulent (R) strain into mouse = mouse survives Step 2 ❑ Boiling of S strain culture + inject heat-killed S cells into mouse = mouse survives ❑ Boiling of S strain culture + inject heat-killed S cells + live R strain = death  DNA: The Genetic Material Frederick Griffith (1928) ❑ Experimental design  DNA: The Genetic Material Frederick Griffith (1928) ❑ Via some mechanism, the cellular debris from the S strain was able to convert the R strain into the virulent form (Rà S strain) ❑ This process is called transformation. Modification of a genome via external application of DNA originating from a separate genotype.  DNA: The Genetic Material Taking these experiments a step further… ❑ Question: What was the molecule/substance responsible for carrying this virulence trait? ❑ Obviously some cellular component from the S strain had stimulated a change in the organism (R strain), and therefore whatever that component was would be a likely candidate for hereditary material.  DNA: The Genetic Material Taking these experiments a step further… ❑ Avery, MacLeod, & McCarty (1944) Experimental design: ❑ Destroy all the major categories of chemicals in the extracts of the dead (S) cells. ❑ See figure on next page for experimental design (note: for each experimental condition, only one category of chemicals would be destroyed) Example: RNase added to heat-killed sample à breakdown of RNA à inject into mouse à DEATH Protease added to heat-killed sample à breakdown of proteins à inject into mouse à DEATH DNase added to heat-killed sample à breakdown of DNA à inject into mouseà SURVIVES Taken together, these experiments strongly implicated DNA as the molecule that carries hereditary information.  DNA: The Genetic Material ❑ Avery, MacLeod, & McCarty (1944) Experimental design: ❑ Destroy all the major categories of chemicals in the extracts of the heat killed (S) cells.  DNA: The Genetic Material Hershey – Chase Experiment ❑ Alfred Hershey and Martha Chase (1952) ❑ Experiments utilized a bacteriophage (T2 virus) and E. coli (host). T2 phage E. Coli with attached phages  DNA: The Genetic Material Hershey – Chase Experiment ❑ At the time, it was understood that the T2 virus was injecting a substance into the host that carried information for the synthesis and assembly of new T2 phage particles. ❑ Phage structure is relatively simple Mostly protein (coat) + DNA  DNA: The Genetic Material Hershey – Chase Experiment ❑ Experimental design: Label DNA and protein components of T2 phage separately ❑ Phosphorus - 32P – found in DNA, but not in proteins ❑ Sulfur – 35S – found in proteins, but not in DNA ❑ Infected two separate E. coli cultures with: 1. 32P-labeled (DNA) T2 virus 2. 35S-labeled (protein) T2 virus  DNA: The Genetic Material Hershey – Chase Experiment ❑ Experimental design: Label DNA and protein components of T2 phage separately ❑ Infected two separate E. coli cultures with: 1. 32P-labeled (DNA) T2 virus 2. 35S-labeled (protein) T2 virus ❑ Allow for sufficient time for infection to take place ❑ Agitate with blender to cause disassociation of phage and bacteria ❑ Separate phage “ghosts” (empty carcasses with no DNA) from bacterial cells (via centrifugation).  DNA: The Genetic Material Hershey – Chase Experiment ❑ Found that In the 32P labeled phages à most of the radioactivity was associated with the bacterial cell fraction. ❑ DNA entered the host cell Therefore, DNA was molecule transmitting inheritance information In the 35S labeled phages à most of the radioactivity associated with the phage “ghost” fraction. ❑ Protein did not enter the host cell * Griffith’s experiments + MacLeod/McCarty experiments + Hershey/Chase experiments = strong evidence that DNA is hereditary material  DNA Structure Prior to the current understanding of DNA structure, three key properties for DNA were assumed (pre-Watson & Crick): 1. DNA must allow for successful replication of the genetic material at every cell division 2. DNA must encode all the information need for the assembly of proteins expressed by an organism 3. DNA must be able to change (mutations must be possible). ❑ Also, the structure must be relatively stable as well so that organisms can rely on its encoded information.  DNA Structure What information was available prior to Watson and Crick’s discovery of the double helix (1953)? A Structure for Deoxyribose Nucleic Acid Watson J.D. and Crick F.H.C. Nature 171, 737-738 (1953) 1. Building blocks 2. Chargaff’s base composition rules 3. X-ray diffraction data (Rosalind Franklin)  DNA Structure Building blocks of DNA ❑ As a chemical, DNA is really quite simple Three chemical components: 1. Phosphate 2. Deoxyribose (sugar) 3. Nitrogenous base (four different types) i. Adenine ii. Guanine iii. Cytosine iv. Thymine  DNA Structure Building blocks of DNA  DNA Structure Building blocks of DNA ❑ Important: The carbon atoms in the nitrogenous bases and in the sugar molecule are assigned numbers (reference) Sugar group numbers are followed by a prime symbol (1’, 2’, 3’, 4’, and 5’) Sugar is called deoxyribose due to the hydrogen (H) at the 2’ position ❑ Ribose typically has hydroxyl group (- OH) at the 2’ position  DNA Structure Building blocks of DNA ❑ Two if the bases (adenine and guanine) have a double ring structure. These are referred to as purine bases ❑ Cytosine and thymine have single ring structures. These are referred to as pyrimidine bases.  DNA Structure Two chains of the double helix have complementary sequences ❑ Structure of the double helix is due to both shape and hydrogen- bonding properties between A-T & C-G Adenine – Thymine (2 hydrogen bonds) Guanine – Cytosine (3 hydrogen bonds) Exocyclic amino group at C6 on A forms Exocyclic amino group at C2 on G forms hydrogen bond with carbonyl at C4 on T hydrogen bond with carbonyl at C2 on C N1 of A froms hydrogen bond with N3 of T Hydrogen bond forms between N1 of G and N3 of C Hydrogen bond forms between carbonyl at 29 C6 of G and exocyclic amino group of C4 of C  DNA Structure Chargaff’s rules of base composition. 1. The total number of pyrimidine nucleotides (T + C) always equals the total number of purine nucleotides (A + G) 2. Amount of T = amount of A; amount of C = amount of G ❑ Note: A + T not always = G + C. Ratio varies among different organisms.  DNA Structure X-ray diffraction ❑ Rosalind Franklin Her data was critical in the development of the Watson-Crick model for DNA structure. ❑ Data analysis suggested that DNA had two parts (long, skinny) that run parallel to each other. DNA Structure  DNA Structure Watson & Crick Model for DNA ❑ Published findings in the journal Nature (1953) A Structure for Deoxyribose Nucleic Acid Watson J.D. and Crick F.H.C. Nature 171, 737-738 (1953) ❑ Suggested a structure for deoxyribose nucleic acid (D.N.A.) We now abbreviate it as DNA (deoxyribonucleic acid) James Watson – American molecular biologist Francis Crick – British molecular biologist  DNA Structure Watson & Crick Model for DNA ❑ Composed of two side-by-side chains of nucleotides Two strands twist into shape of a double helix ❑ These two strands are held in this helical structure via hydrogen bonds (between the nitrogenous bases) In the analogy of DNA resembling a spiral stair case, think of the hydrogen bonds as the “steps”  DNA Is a Double Helix DNA Conformation ❑ DNA is typically a right-handed double helix (B-DNA). ❑ Periodicity Each base pair is twisted from the previous one by ~36° ❑ Thus, it takes approximately 10 base pairs to go completely around the helix. Actually there are 10.5 bases per helical turn of DNA One helical turn = 34 Å (angstrom) note: 1 Å = 0.1 nm 35  DNA Is a Double Helix ❑ Double helix structure gives rise to two distinct sizes of grooves that are not equal in size. 1. Major groove = 12 Å (wide) 2. Minor groove = 22 Å (wide) ❑ Most of the DNA-protein associations occur in major grooves 36  DNA Is a Double Helix Major and minor grooves ❑ In these grooves, the edges of the base pairs (A-T; G-C) are exposed. ❑ Look at the distinct “fingerprint” of (note here that A does NOT indicate an adenine): A = hydrogen bond acceptor D = hydrogen bond donor H = Nonpolar hydrogen M = methyl group 37  DNA Is a Double Helix Major and minor grooves ❑ Distinct “code” for protein association. ADAM = A – T MADA = T – A AADH = G – C HDAA = C – C AHA = A –T or T – A ADA = C- G or G - C 38  DNA Is a Double Helix Major and minor grooves Distinct “code” for protein association. ❑ ADAM = A - T Major groove - Rich in information regarding ❑ MADA = T – A protein interaction ❑ AADH = G – C ❑ HDAA = C – C Not as much can be discerned from reading this…less ❑ AHA = A –T or T – A specific for protein interactions ❑ ADA = C- G or G - C 39  DNA Is a Double Helix Major groove ❑ Pattern of hydrogen bonds (along with methyl groups) allows proteins to recognize specific regions of DNA Without having to open and close sections of DNA (i.e, scanning for correct activation site). ❑ ADAM = A - T ❑ MADA = T – A ❑ AADH = G – C Example: NR5A1 recognition site: ❑ HDAA = C – C …AGGTCA 40  DNA Structure Watson & Crick Model for DNA ❑ “Backbone” of each strand made up of alternating phosphate + deoxyribose sugar units (sugar-phosphate backbone) ❑ Specifically, a phosphodiester linkage connects the 5’-carbon of one deoxyribose to the 3’-carbon of another deoxyribose The sugar-phosphate backbone is said to have 5’-to-3’ polarity ❑ This is key to understanding how DNA replicates. New DNA strands are synthesized in 5’ to 3’ direction  DNA Structure Watson & Crick Model for DNA ❑ In the double helix structure, the two sugar-phosphate backbones run in opposite (antiparallel) direction. ❑ As base pairing occurs between two strands, the base pairs stack on top of one another. This adds stability to the DNA molecule (excludes water molecules from the spaces between base pairs  DNA Structure Watson & Crick Model for DNA ❑ Double helix structure gives rise to two distinct sizes of grooves 1. Major groove 2. Minor groove ❑ Most of the DNA-protein associations occur in major grooves  DNA Structure Watson & Crick Model for DNA ❑ Base pairing rule Watson and Crick realized that the radius of the double helix could be explained if a purine and a pyrimidine always pair, Concluded that: ❑ G (on one strand) always pairs with C (on the complementary strand) ❑ A always pairs with T  DNA Structure Watson & Crick Model for DNA ❑ The G-C pair has three hydrogen bonds ❑ The A-T pair has two hydrogen bonds G-C pair more stable (than A-T) High G-C content requires higher temperatures to separate (melt) two strands of DNA (compared to high A-T content)  DNA Structure Watson & Crick Model for DNA ❑ Awarded Nobel Prize in 1962 By this point, Rosalind Franklin had died of cancer (not awarded posthumously) ❑ Their proposed model fulfilled three requirements for the proposed genetic material.  DNA Replication Watson & Crick Model for DNA ❑ Three requirements: Sequence of nucleotides may encode information about protein assembly (genetic code). Changing a base in DNA (mutation) could change the way (code) in which protein is synthesized Possible method of replication ❑ “It has not escaped our notice that the specific pairing…suggests a possible copying mechanism…”  DNA Replication The mechanism to which Watson and Crick eluded was that of semiconservative replication Each strand of the double helix would serve as a template for synthesis of a new strand.  DNA Replication Zipper analogy (double helix) ❑ Unzips at one end ❑ Unzipping (unwinding) of strands ❑ Expose bases on each of the two strands ❑ Exposed bases can potentially pair with free nucleotides ❑ Strict pairing requirements (only pair with complementary base) A-T G-C  DNA Replication Therefore, each of the two single strands act as templates. Direct the assembly of complementary nucleotides to reform a double helix structure identical to the original. ❑ For each newly synthesized double helix, DNA contains one parental strand + one newly synthesized strand This mechanism is called semiconservative replication  DNA Replication Another predication of the Watson & Crick model… ❑ Replication fork Location in DNA at which the double helix is unwound to produce two single strands ❑ Remember: these can then function as templates for new strand synthesis Bacterial chromosome  DNA Replication DNA synthesis enzyme(s) ❑ Although previously suspected, it was not until 1959 that researchers found evidence of specific enzymes involved in replication ❑ Arthur Kornberg – isolated DNA polymerase from E. coli. DNA polymerase  DNA Replication DNA polymerase ❑ Enzyme that adds deoxyribonucleotides to the 3’ end (OH group) of a growing nucleotide chain Substrates for this enzyme: dATP, dCTP, dGTP, dTTP ❑ Now know of five different DNA polymerase enzymes in E. coli.  DNA Replication Reaction catalyzed by DNA polymerase  DNA Replication DNA polymerase ❑ This first isolated polymerase called DNA polymerase I (pol I) Has three activities: 1. Polymerase activity; catalyzes strand elongation in 5’ to 3’ direction. 2. 3’ – 5’ exonuclease activity; removes mismatched bases (proof reading ability). 3. 5’ – 3’ exonuclease activity; degrades double-stranded segments of DNA. ❑ Side note: Known that DNA polymerase III (pol III) catalyzes DNA synthesis at the replication fork  DNA Replication Overview of DNA replication ❑ For our introduction to this concept, we will begin with the basic mechanisms of replication in bacteria. E. coli  DNA Replication Overview of DNA replication ❑ Let’s start at the replication fork DNA pol III moves forward as the double helix is continuously unwinding (just ahead of this enzyme) ❑ This unwinding functions to expose single strands of DNA for pairing with complementary nucleotides (as discussed earlier).  DNA Replication Overview of DNA replication ❑ Something we must consider…. As nucleotides are added to the growing DNA strand, they are added ONLY at the 3’ end of the growing strand. (i.e., 5’ to 3’ direction of synthesis) Therefore, only one of the two antiparallel strands serves as a template for replication in the direction of unwinding.  DNA Replication Overview of DNA replication ❑ Something we must consider…. Synthesis of this strand (in the direction of unwinding) is a smooth continuous process. This newly synthesized strand is properly referred to as the leading strand.  DNA Replication Overview of DNA replication ❑ Something we must consider…. Synthesis on the other template also occurs at the end of the 3’ growing strand. However this synthesis is in the “wrong” direction. ❑ Occurs away from the unwinding at the replication fork  DNA Replication Overview of DNA replication ❑ Something we must consider…. Synthesis of this strand (in the “wrong” direction) occurs in short segments (not continuous) Extends for some period of time, then DNA pol III moves back toward the replication fork ❑ New segment is then synthesized using the newly exposed template section  DNA Replication Overview of DNA replication ❑ Something we must consider…. Each fragment is typically 1000-2000 nucleotides in length. These short stretches of newly synthesized DNA are called Okazaki fragments. After the production of Okazaki fragments, the process of replication is still not complete for this lagging strand. Lagging strand Leading strand  DNA Replication Overview of DNA replication ❑ For both the leading and lagging strands DNA polymerase functions to extend a nucleotide chain, but it cannot START a new chain! The leading strand and each Okazaki fragment must be initiated by a primer. ❑ Short nucleotide chain that binds to template (acts as a start site for DNA polymerase. Lagging strand Leading strand  DNA Replication Overview of DNA replication ❑ For both the leading and lagging strands These short chain nucleotides (primers) are synthesized by a primosome. Specifically, the enzyme primase (RNA polymerase) is the major functional unit of the primosome. ❑ Primase synthesizes short RNA fragments (8-12 nucleotides) that are complementary to specific regions of the DNA For the leading strand, only one initial primer is needed. For the lagging strand, each individual Okazaki fragment needs its own primer Lagging strand Leading strand  DNA Replication Overview of DNA replication ❑ Other polymerases and enzymes In reference to the lagging strand, there are a number of “touch ups” that are necessary before synthesis can be completed. Lagging strand Leading strand  DNA Replication Overview of DNA replication ❑ Other polymerases and enzymes DNA pol I - removes the RNA primers and fills in the resulting gaps (8-12 nucleotides each) with DNA. ❑ (This is true for both the leading and lagging strand, it is just that there are more gaps to fill for the lagging strand) DNA ligase – functions to join the 3’ end of the gap-filling DNA to the 5’ end of the Okazaki fragment.  DNA Replication Overview of DNA replication ❑ Other polymerases and enzymes DNA ligase – functions to join the 3’ end of the gap-filling DNA to the 5’ end of the Okazaki fragment.  DNA Replication Overview of DNA replication ❑ DNA replication accuracy ❑ Often referred to as fidelity. ❑ Typically, one error per 1X1010 nucleotides added. ❑ 3’ to 5’ exonuclease activity of DNA polymerase (proof-reading) allows for this high level of fidelity  DNA Replication The replisome ❑ Large nucleoprotein complex that coordinates the activities at the replication fork  DNA Replication The replisome ❑ Components of the replisome (for E. coli) Catalytic core – DNA pol III ❑ Part of a much larger complex called the pol III holoenzyme. Has two catalytic cores + accessory proteins One catalytic core (DNA pol III) responsible for leading strand synthesis The other catalytic core (DNA pol III) responsible for lagging strand synthesis.  DNA Replication The replisome ❑ Components of the replisome (for E. coli) Important accessory protein is called the β clamp. ❑ Keeps DNA pol III attached to the DNA molecule ❑ Without this clamp, would add ~10 nucleotides, then fall off; with clamp, adds tens of thousands of nucleotides (processive enzyme).  DNA Replication The replisome ❑ Components of the replisome (for E. coli) Important accessory protein is called the β clamp. ❑ Primase (RNA pol) is NOT attached to the clamp; explains why only ~10 nucleotides are linked for the RNA primer (primase = distributive enzyme)  DNA Replication The replisome ❑ Components of the replisome (for E. coli) The replisome also contains two classes of proteins that open up the helix and prevent overwinding of DNA. 1. Helicases 2. Topoisomerases  DNA Replication The replisome ❑ Components of the replisome (for E. coli) Helicases – disrupt hydrogen bonds between bases; unzips the double helix ❑ Unwound DNA further stabilized by single stranded binding proteins (SSB). Prevent immediate reformation of double helix  DNA Replication The replisome ❑ Components of the replisome (for E. coli) Topoisomerases – cut DNA strand(s) to relax supercoiled regions. Once relaxed, functions to rejoin the DNA strand(s) ❑ Ex. DNA gyrase  DNA Replication The replisome ❑ Assembling the replisome Begins at precise sites on the chromosome (origins) ❑ Ori (origin) In E. coli, begins at fixed site called oriC.  DNA Replication Replication in eukaryotic organisms ❑ Also uses semi conservative mechanisms with leading and lagging strands ❑ However, with increasing complexity of the organism, the number of replisome components also increases ❑ 13 components for E. coli replisome; at least 27 components for mammalian replisomes  DNA Replication Eukaryotic replisome ❑ Due to the presence of nucleosomes (organization units of chromosomes), the replisome must copy the parental strands, plus it must disassemble the nucleosomes and then reassemble them in the daughter molecules  DNA Replication Eukaryotic origins of replication ❑ Approximately 400 origins dispersed throughout the 16 chromosomes of yeast ❑ Estimated to be thousands for origins among the chromosomes in the nucleus of a human cell. ❑ In eukaryotes, replication proceeds in both directions from multiple points of origin for each chromosome  DNA replication Telomeres ❑ Located at the tips of the chromosomes ❑ Act to “seal” the ends of chromosomes Form loop structure at the ends of each chromosome Helps stabilize the chromosome end ❑ DNA synthesis enzyme (polymerase) cannot replicate new strands all the way to the end of each chromosome. Quick loss of genetic material.  Chromosomes Telomeres ❑ Loss of telomeres have detrimental effects. ❑ Results in an increase in mutations rates in these cells ❑ Telomerase – enzyme that functions in the synthesis and maintenance of these loops (telomeres) at chromosomes ends.  DNA Replication Telomeres