AP Biology Chapter 13 + 14

DNA Structure

• Double helix

  • Made up of 2 adjacent polynucleotide strands wound around an imaginary axis into a spiral shape

• DNA is a polymer of nucleotides 

• Three components: 

• nitrogenous bases (nitrogen-containing)

  • Adenine (A)

  • Thymine (T)

  • Guanine (G) 

  • Cytosine (C)

• Pentose group 

  • DNA → deoxyribose 

  • Pentose sugar (5 carbons) 

    • The phosphate group is attached to the 5’ carbon 

    • Hydroxide (OH-) is attached to the 3’ carbon 

    • Nitrogenous bases are attached to the 1’ carbon 

• Phosphate group

• Sugaphosphate backbone = outside the DNA molecule 

  • Phosphodiester bonds hold nucleotides together in the backbone 

  • Negatively charged phosphate groups facing the aqueous surroundings 

  • Hydrophobic nitrogenous bases = hidden 

  • Helix makes one full turn every 3.4 nanometers (10 bp) 

    • Each base pair is stacked 0.34 nm apart

• Hydrogen bonds between bases hold strands together 

• Vanderwall's interactions between stacked base pairs hold the molecule together

Antiparallel + Semi conservative

• Strands are antiparallel → oriented in opposite directions 

• Two strands are also complementary 

  • Nucleotides line up along the template strand according to base pairing rules 

• Semiconservative: each of the two daughter molecules has one old strand (from parental molecule) and one newly made strand

Base Pairings

• Specific base pairings

  • A → T (Apple Tree) 

  • G → C (Garbage Can)

  • Because A and G are purines (two organic rings) 

    • Cytosine and thymine pyrimidines (single ring) 

  • Purine with pyrimidine is the only combination that results in a uniform diameter for the double helix

    • Additionally → each base has a chemical side group that forms hydrogen bonds with its partner

      • A forms 2 bonds with T and only T 

      • G forms 3 bonds with C and only C 

• Ratios of the base pairs

  • amount of adenine = amount of thymine 

    • always paired together, so where one shows up, the other does as well

  • Amount of guanine = amount of cytosine (always exactly equal)

DNA replication

• DNA replication occurs during the S phase of interphase 

  • Semiconservative replication 

    • Prokaryotes → one origin of replication

• Multiple origins of replication on each strand → stretches of DNA that have a specific nucleotide sequence 

  • Proteins recognize the sequence and attach to the DNA, separating the strand to form replication bubbles 

    • Multiple replication bubbles → allows for efficiency 

    • At the end of each bubble is a replication fork

• Replication goes in both directions from each origin 

Enzymes

• Helicase enzyme untwists the double helix at the fork → separating the parental strands to become template strands 

• Single-strand binding proteins → bind to DNA to keep it from repairing 

  • Untwisting causes tighter twisting and strain ahead of the replication form 

• Topoisomerase: relieves strain by breaking, swiveling, and rejoining DNA strands

  • stops unraveled DNA from supercoiling → remains split 

• Primase: makes RNA 

• DNA Polymerase: building complementary DNA nucleotides

  • Multiple different kinds → the enzyme removes the RNA primer, and adds more nucleotide bases 

    • Adding is done via a condensation reaction 

      • Two phosphate groups are lost as a molecule of pyrophosphate 

        • Hydrolysis of pyrophosphate to two molecules of phosphate is a coupled exergonic reaction → drives polymerization reaction

• Ligase: links fragments and sugar phosphate backbones together 

  • Creates a continuous strand 

Leading + lagging strand

• DNA is built in a 5’ → 3’ direction (every nucleotide has a 5’ end next to a 3’ end)

  • Enzymes that synthesize DNA cannot start without an existing chain that has been base-paired with the template strand 

    • The initial chain is a short stretch of RNA primer (typically 5-10 bases long)

      • DNA starts from the 3’ end of the RNA primer

  • leading strand → strand built into the fork  

    • Only needs one primer to synthesize the whole strand 

  • Lagging strand → built away from the fork

    • Built in segments called Okazaki fragments 

    • Takes longer 

Error Repair

• Errors in the completed DNA molecule amount to only 1 in 10 billion 

• DNA polymerase proofreads newly made DNA against its template, replacing any incorrect nucleotides 

  • Immediate change as soon as the bases are added

    • Once an error is found → polymerase removes the nucleotide and resumes synthesis 

• Mismatch repair: after DNA has been copied 

  • Other enzymes remove and replace incorrectly placed nucleotides from replication errors 

    • double-check and correct base parings

  • A hereditary defect in one such enzyme is associated with a form of colon cancer 

    • The defect allows cancer-causing errors to accumulate in DNA faster than normal 

• Nucleotide excision repair (mechanism used for incorrect base pairing)

  • The segment of the strand containing damage is cut out by a nuclease (enzyme)

    • The gap is filled using the undamaged strand as a template 

      • Using DNA ligase + polymerase 

• Permanent damage in the DNA sequence = mutation

Prokaryotes

• Prokaryotic organisms (bacteria)  → replication is much faster

  • Single, double-stranded circular DNA molecule → associated with small amounts of proteins 

  • Certain proteins cause the chromosome to supercoil, densely packing it so it fills only one part of the cell 

    • The region is called the nucleoid 

Eukaryotes

• Multiple chromosomes → contain linear DNA molecules with large numbers of proteins 

• Chromatin: a complex of DNA and protein found in the nucleus of eukaryotic cells 

• Chromosomes fit into the nucleus through an elaborate multilevel system of packing 

  • Proteins called histones are responsible for the first level of DNA packing in chromatin 

  • Four types of histones are common in chromatin 

  • A nucleosome consists of DNA wound twice around a protein core of 8 histones → two of each of the main histone types 

• Chromatin undergoes a striking change in the degree of packing during the course of the cell cycle

  • Metaphase: Chromosomes are the most condensed

  • Prepping for Mitosis 

    • Chromatin condenses (coils), forming short and thick chromosomes 

DNA cloning

• DNA cloning

• method for prepping well-defined segments of DNA in multiple identical copies 

• Most methods for cloning pieces of DNA in the laboratory

• Use of bacteria → many have plasmids 

  • small circular DNA molecules that replicate separately from the bacterial chromosome 

• To clone pieces of DNA → Researchers obtain a plasmid and insert DNA from another source into it

  • Called horizontal gene transfer → move plasmids to transfer DNA

  • The resulting plasmid is called recombinant DNA (molecule with DNA from two different sources) 

• Plasmid is returned to the bacterial cell → recombinant bacterium 

  • A single cell reproduces through cell divisions to form a clone of cells 

• Plasmids act as a cloning vector → A DNA molecule that carries foreign DNA into a cell and is replicated there

  • Makes copies or amplifies a particular gene to produce a protein product from it 

  • Used to create new bacteria, proteins (insulin), or more that are beneficial

    • Also used to study genes

Restriction enzymes

• Genetic engineering relies on these enzymes that cut DNA molecules at a limited number of specific locations 

• Each one recognizes a particular sequence, or restriction site 

  • Cuts both strands at a specific point 

Gel Electrophoresis

• uses a gel made of a polymer as a sieve to separate a mixture of nucleic acid fragments by length 

• DNA is negatively charged → moves to the positive end 

  • Shorter molecules move faster through the cell 

  • Separates base segments and gene segments 

  • Helps compare base strands of DNA 

  • And identify strands 

PCR

• (Polymerase chain reaction) → produces many copies of a specific target segment of DNA 

  • A three-step cycle brings about a chain reaction that produces an exponentially growing population of identical DNA molecules 

    • Step 1: heated to denature the strands 

    • Step 2: cooled to allow annealing (hybridization) of a short single-stranded DNA primer 

    • Step 3: DNA polymerase extends the primers 

      • Use a heat-stable DNA polymerase enzyme called Taq polymerase 

        • From bacterial species that live in hot temps 

  • PCR is used to provide DNA fragments for cloning 

    • PCR primers include a restriction site at each end that matches the site in the cloning vector 

      • Cut and ligated together

    • After the gene is cloned → DNA sequencing 

CRISPR-Cas9 system 

• Cas9 is a nuclease (enzyme) that cuts double-stranded DNA molecules as directed by a guide RNA that is complementary to a target gene

  • When given a restriction enzyme → Cas9 will cut any sequence where it is directed 

• Researchers use this system to knock out (disable) a given gene to determine its function

  • Specifically used to correct a genetic defect that causes sickle cell disease

One gene = One polypeptide

• Beadle + Edward Tatum → experimented with bread mold Neurospora crassa 

  • Haploid, and modest food requirements 

  • Able to create mutants by observing the metabolic pathways and steps 

• discovered that one gene = one enzyme hypothesis 

  • The function of a gene dictates the production of a specific enzyme

• Made revisions to the theory as discoveries surfaced 

• Not all proteins are enzymes → one gene = one protein?

  • However, many proteins are constructed from more than one polypeptide chain 

  • Additionally, many genes can code for different proteins (alternative splicing) 

  

RNA

• Ribose instead of deoxyribose 

• Has a nitrogenous base of uracil instead of thymine 

• Typically single-stranded (not double helix)

• Nucleic acids have genetic information in nucleotides

  • Proteins contain information in amino acids → same info, different way

Ribosomes

• large and small subunit → each made up of proteins and one or more rRNA (ribosomal RNA)

  • In eukaryotes, subunits are made in the nucleolus 

  • Subunits are exported via nuclear pores → both join to form a functional ribosome when attached to an mRNA molecule

rRNA and mRNA

• rRNA (ribosomal RNA → most abundant)

  • Helps assemble ribosomes 

  • Reads mRNA to create a polypeptide chain 

• mRNA (messenger RNA) 

  • Complementary copy of DNA 

  • Carries a genetic message from the DNA to the ribosomes

tRNA

• Transfer amino acids from the cytoplasmic pool to the mRNA/ribosome 

  • Cell keeps cytoplasm shocked with all 20 amino acids → takes from surrounding solutions, or synthesizes them from other compounds

• Structure:

  • A single RNA strand with stretches of complementary bases → hydrogen bonds, and forms a 3D structure

    • 3’ end acts as an attachment site for amino acids 

    • The other loop includes the anticodon nucleotide triplet that base pairs to a specific mRNA codon 

  • Enzymes called aminoacyl-tRNA synthetases match tRNA and amino acids

    • Active site only matches one specific amino acid + tRNA 

    • 20 different synthetases

    • Catalyzes the attachment of the amino acid to its tRNA → then releases it 

  • P Site → where the tRNA holding the growing polypeptide chain is attached to  

  • A site → tRNA with the next amino acid

    • After done → exit from the E site 

• Some tRNAs can bind to more than one codon 

  • Flexible base pairing → wobble 

Transcription

• Synthesis of RNA using information in DNA 

• The DNA strand (template strand) provides for assembling the complementary RNA strand 

  • RNA polymerase pries the two strands of DNA apart 

    • Binds to a specific nucleotide sequence to initiate transcription → called promoter (orients the enzyme)

  • RNA polymerase only works in a 3’ → 5’ direction, joining RNA nucleotides with the complementary DNA template strand

    • Can start from scratch → no primer needed

    • Use transcription factors to mediate the process (helps RNA polymerase bind too)

  • At the end → RNA polymerase transcribes a sequence on the strand called the polyadenylation signal sequence 

    • After many bases of this sequence → proteins cut the transcript free from the polymerase, releasing the pre-MRNA

• Before it goes to ribosomes → must be modified to produce functional mRNA

  • Editing only happens in eukaryotes

Translation

•  synthesis of a polypeptide by translating the information  the mRNA molecule  

  • Site of translation = ribosomes (facilitates the linking of amino acids into polypeptide chains)

• Translator = tRNA (transfer RNA) → brings amino acids to ribosome

• The small ribosomal subunit binds to the 5’ cap of the mRNA, scanning downstream until it reaches AUG 

  • Initiator tRNA (with met) is brought over using anticodons → hydrogen bonds to the chain at the P site 

• Amino acids are added one by one to the previous amino acid at the C-terminus (carboxyl end) of the growing chain (to form polypeptide bonds)

  • Also removes amino acids from the tRNA 

• One stop codon is reached → release factor binds to stop codon in the A site 

  • Causes hydrolysis (breaks a bond using water) 

  • Releases peptide 

• Hydrolysis of two GTP molecules breaks apart the ribosome after 

RNA processing

• both ends are altered 

  • 5’ end receives a 5’ cap (modified form of guanine nucleotide) 

  • 3’ end receives more bases of Adenine nucleotides → forming poly-A tail

    • Both facilitate the export of mRNA from the nucleus 

    • Protect mRNA from degradation by hydrolytic enzymes 

    • Help ribosomes attach to the 5’ end of the mRNA in the cytoplasm

RNA splicing

• introns of the RNA molecule are removed, and the remaining sections (exons) are reconnected (join together)

  • Using sNRPS proteins on spliceosomes (a large complex of proteins and small RNAs) to release introns for rapid degradation and join together exons

• Noncoding segments that lie between coding regions → intervening sequences (introns) 

• Other regions → exons 

  • Forms an mRNA molecule with a continuous coding sequence 

• Alternative RNA splicing 

  • Genes create different polypeptides depending on which segments are considered exons

    • An intron for one version of a gene may not be an intron for a different gene 

      • Allows genetic variability

Euks + proks

• Both processes occur in all organisms 

• In bacteria → no nuclei 

  • The membrane does not separate DNA and mRNA from ribosomes 

    • Lack of compartmentalization → means translation can begin while transcription is occurring 

      • Do not need to be modified

• In eukaryotes → nuclear envelope separates the processes

  • All DNA is in the nucleus

  • Transcription occurs in the nucleus → mRNA MUST be transferred to the cytoplasm for translation 

    • But also allows multiple different kinds of proteins through introns + exons

Codons + reading frame

• Codons: triplets of mRNA nucleotide bases that code for all of the amino acids

  • 64 codons → 61 code for 20 amino acids 

    • Other 3 are stop signals or termination codons

      • UAA, UAG, UGA

  • AUG (Met) → start codon 

• Reading frame: triplet groupings of ribonucleotides using protein-synthesizing machinery 

Mutations

• Point mutations → changes in a single nucleotide pair of genes

  • If it occurs in a gamete → only way it can be transferred 

    • Ex: SCD 

• Small-scale mutations 

  • Single nucleotide-pair substitutions 

    • Replacement of one nucleotide and its partner with another pair of nucleotides

      • Some have no effect (redundancy of the genetic code) → silent mutation

      • Some change one amino acid to another → missense mutations 

        • Usually have little effect on the protein 

      • Can change an amino acid into a stop codon → nonsense mutations 

        • Premature end to translation → nonfunctional protein 

  • Nucleotide pair insertions and deletions

    • Additions or losses of nucleotide pairs in a gene 

    • Causes a major change in the protein 

    • Frameshift mutation → when insertion/deletion is not a multiple of 3

      • Usually leads to misense mutations and eventually a nonsense mutation

      • Usually non-functional unless it occurs at the end of a protein 

How do errors occur

• Errors during DNA replication, recombination, or repair  → Spontaneous mutations 

• Physical and chemical agents (mutagens) interact with DNA to cause mutations 

  • Researchers have developed methods to test the mutagenic activity of chemicals 

  • Most cancer-causing chemicals (carcinogens) are mutagenic, and the converse is true