Molecular genetics: focuses on the macromolecules that carry genetic information- DNA, RNA, proteins

For DNA to be widely accepted as genetic material, it must:

1. be present in chromosomes in the cell nucleus

2. double during the S phase of the cell cycle

3. be twice as abundant in diploid cells than in haploid cells

4. have the same transmission pattern as the genetic information it carries

Frederick Griffith: discovered transformation- the ability of organisms to take up DNA from the environment

Alfred Hershey and Martha Chase: identified DNA as the genetic material of the bacteriophage

DNA’s 3D Structure:

James Watson and Francis Crick: model of DNA structure

• DNA has a double helix structure with nitrogenous bases oriented inward toward one another and a sugar phosphate backbone on the outside

• Adenine and thymine pair via 2 hydrogen bonds

• Cytosine and guanine pair via 3 hydrogen bonds

DNA replication is semiconservative

Watson and Crick’s conclusions were based on three different sets of evidence:

1. Chargaff’s rule:

   • DNA is a polymer with: nitrogenous base, pentose sugar, phosphate group

   • noticed DNA composition varies among species

   • amount of adenine = thymine and cytosine = guanine

   • purines and pyrimidines are found in equal abundance is called Chargaff’s rule

2. Rosalind Franklin:

   • x-ray crystallography and diffraction

   • DNA is a helix spiral

3. Chemistry models:

   • ball and stick models for structure and function

Important features of DNA- Watson & Crick:

• DNA is double stranded helix with a uniform diameter

• The two DNA strands run antiparallel

• The outer edges of the nitrogenous bases are exposed in major and minor grooves, making them available for hydrogen bonding with proteins

Models of DNA:

• Conservative model: the parental strand remains intact, new strands are completely new

• Semi-conservative model: one of the strands is a new strand, one is old

• Dispersive model: each resulting strand of both daughter DNA molecules has both old and new parts

DNA Replication:

1. unwinding the double helix, forming two template strands

2. using the template strands to form daughter strands through complementary base pairing with new nucleotides

• DNA polymerase is processive: works fast, catalyzes sequential polymerization reactions every time it binds to DNA

• antiparallel lines = 3’ and 5’ end

• phosphodiester bonds: link the 3’ end of one sugar to the 5’ end of the next sugar

• DNA polymerase adds nucleotides on the growing strand of DNA in the 5’ → 3’ direction

  • leading strand: DNA synthesis occurs in one continuous motion as the replication fork expands

  • lagging strand: synthesis proceeds away, Okazaki fragments

  • DNA ligase joins okazaki fragments together

Proofreading and Mismatch pair:

• Proofreading: DNA polymerase’s capability to stop DNA synthesis if the wrong nucleotide is entered

• Mismatch pair: use of special enzymes to fix incorrectly paired nucleotides after DNA replication has occurred

• make the final error rate much smaller

The End Replication Problem & Telomeres:

• End-replication problem: the fact that there is no way to replace the RNA primer at the 5’ end of the daughter strand that is complementary to the lagging strand

• in every round of replication, daughter DNA molecules would get shorter and shorter

• telomeres get shorter and shorter, after about 20-30 cell divisions, too short for replication

• Telomeres: nucleotide sequences that repeat at the end of chromosomes, do not have genes, buffer, prevent erosion of important genes and improper joining together of damaged DNA by normal DNA repair mechanisms

• Telomerase: an enzyme that catalyzes the lengthening of telomeres in the germ line and some cancer cells

  • does not work in normal somatic cells

  • germline = reproductive cells

  • related to aging: older people have shorter telomeres, more susceptible to diseases and health problems

Enzymes:

Mutations:

• Mutation: heritable change to DNA, ultimate source of all genetic variation

• Somatic mutation: passed down through mitosis, only affects singular individual

• Germline mutation: passed down during sexual reproduction, heritable

• Mutations create new genetic variation by changing the existing DNA

• Most mutations are neutral or deleterious, extremely rare for it to be beneficial

Causes of Mutations:

• spontaneous mutations: no outside influence from the environment, permanent

  • ex: DNA polymerase makes a replication error that does not get corrected

• induced mutations: caused by a mutagen (outside agent)

  • ex: chemicals, radiation (UV and Xrays) infectious agents (viruses, bacteria), can be natural or man made

Types of Mutations:

• Silent mutations: leave the protein unaffected (majority)

• Loss of function mutations: inhibit the function of the final protein or enzyme, almost always recessive, some result in cancer

• Gain of function mutation: alter or promote the function of a final protein or enzyme, rare, usually dominant, found in cancer cells

• DNA mutation: small scale mutation that operates on just a small portion of the DNA

  • ex: point mutations, frameshift mutations

• Chromosome mutations: change in the structure of a chromosome

Point Mutations:

• Point mutation: occurs when a single nucleotide in a genome changes

• can result from environmental mutagens (toxic chemicals, UV radiation) or from errors in DNA replication

• occurs in coding region of DNA = mRNA transcript is often changes

• Sickle-cell disease: single base substitution that changes from glutamic acid to valine

Base substitutions: one nucleotide is replaced with another nucleotide

• Synonymous base substitutions (silent mutation): changes nucleotide, has no effect on polypeptide

• Non synonymous base substitution: changes DNA sequence AND polypeptide

  1. Missense substitution: changes resulting amino acid, but rest of polypeptide remains intact

  2. Nonsense substitution: introduces premature stop codon (UAA, UAG, UGA) resulting in shortened polypeptide that is not functional

Frameshift Mutations (Base Insertions and Deletions):

• Frameshift mutation: occurs when one or two bases are inserted into a protein-coding sequence and alter the reading frame

• Insertion: addition of extra bases to the DNA sequence

• Deletion: the loss of bases from the DNA sequence

• Adding in bases in multiples of 3 does not cause a frameshift mutation because it adds an entire codon

Chromosome Mutations:

• Chromosome mutation: change in the structure of a chromosome

• Deletion: genes are completely removed from a chromosome

• Duplication: occurs when genes are reproduced and appear together on the same chromosome

• Inversion: occurs when a chromosome breaks and rejoins, leading to segments of genes that are flipped backwards on the chromosome in a 180 degree reversal

• Translocations: crossover between nonhomologous chromosomes rather than homologous

The Causes of Mutations:

• spontaneous mutations: no outside influence from the environment, permanent

  • ex: DNA polymerase makes a replication error that does not get corrected

  • errors in DNA replication

  • improper base pairing (tautomer)

  • chemical reactions that change bases

    • deamination: loss of an amino group

• induced mutations: caused by a mutagen (outside agent)

  • ex: chemicals, radiation (UV and Xrays) infectious agents (viruses, bacteria), can be natural or man made

  • chemicals that alter nucleotide bases

  • chemicals that add other groups to bases

  • ionizing radiation: xrays and gamma rays creates highly reactive free radicals that can break sugar-phosphate bonds

  • uv radiation: thymine-thymine dimer forms a bubble that blocks replication and transcription

Costs and Benefits of Mutations:

Costs:

• mutations can be harmful or lethal

• some mutations can cause cancer or lead to tumors: tumor suppressor cells, oncogenes

Benefits:

• some mutations immediately impact reproductive fitness

• others increase genetic diversity and provide the raw material for evolution

Transcription and Translation:

• Sir Archibald Garrod: linked human diseases with people’s inability to make particular enzymes

• “one gene, one enzyme” - one gene codes for one polypeptide

Central Dogma:

• Central Dogma of molecular biology: flow of genetic information from DNA to RNA to protein

  • transcribed from DNA to RNA and translated from RNA to proteins

  • Transcription: synthesis of RNA from genetic information, one of the strands is a template for the synthesis of mRNA which carries genetic message from the DNA to be carried out in the protein

  • Translation: synthesis of polypeptides from information carried in RNA, occurs on ribosomes where tRNA physically links mRNA and amino acids

  • DNA → pre-MRNA → mRNA → immature polypeptide → final protein

Types of RNA

• messenger RNA: piece of RNA translated at the ribosome

• ribosomal RNA: structural component of ribosomes

• transfer RNA: binds to specific sequences on mRNA, bringing specific amino acids to the growing polypeptide chain

Transcription:

1. requires DNA template, RNA polymerase, nucleoside triphosphates (ATP, GTP, CTP, UTP)

2. Initiation: RNA polymerase binds to the template strand called the promoter sequence and starts to unwind the DNA

3. Elongation: RNA polymerase connects nucleotides that base-pair with the template strand nucleotides, forming RNA. Adds from 5’ → 3’ (can only add at 3’)

4. RNA polymerase does not require a primer

5. Termination: RNA polymerase reaches the terminator sequence, stops transcription and disengages from DNA

• in prokaryotes: mRNA produced immediately translated into protein without further processing

• eukaryotes: transcription results in pre-mRNA, then turns to mRNA, then transported from nucleus to ribosomes for translation

Post-Transcriptional Processing in Eukaryotes:

• pre-mRNA must be enzymatically modified in 3 important ways before being shipped outside the nucleus":

  1. 5’ cap is added to protect transcript from degradation by ribonucleases and serves as a recognition signal for ribosomes. ensures the mRNA remains stable being transported out of the nucleus and while it undergoes translation

  2. poly-A tail (adenine nucleotides) added to the 3’ end of mRNA, protect transcript from degradation by ribonucleases and serves as a recognition signal for ribosomes, and is necessary for export of mRNA to cytoplasm

  3. introns: sequence of nucleotides that gets removed from pre-mRNA and is never translated. introns must be cut out of split genes during RNA splicing. extrons joined together by spliceosomes (snRNPs) to form fully functional mRNA

The Genetic Code:

• 3 bases form a codon of mRNA that codes for a specific amino acid

• 64 possible codons, 20 amino acids

• redundant, but not ambiguous

• nearly universal

• start: AUG

• stop: UAA, UAG, UGA

Translation:

• tRNA provides the link between mRNA and protein by transferring amino acids from the cytoplasm to the ribosome

• each tRNA has an anticodon that is complement to the base mRNA codon

• occurs at the ribosomes: large and small subunit

1. initiation: small subunit binds the mRNA at the 5’ end and binds tRNA at start codon

2. first amino acid synthesized is always MET (methionine)

3. elongation: codon in A-site hydrogen bonds with anticodon of charged tRNA

4. polypeptide in P-site and amino acid in A_site are joined together by peptidyl transferase ribozyme peptide bond

5. termination: protein release factor binds to stop codon in the A-site, releases completed polypeptide in the P-site from the ribosome

• many ribosomes can use the same strand of mRNA at once = produce multiple copies of proteins from a single mRNA quickly

• polypeptide chain is not functional unless it has been folded (enzymatically modified to perform its specific function)

  • folding: chaperone proteins assist in achieving the proper folding of the polypeptide to form secondary and tertiary structures

  • addition of phosphate and sugar groups: phosphorylation and glycosylation

  • removal: certain segments need to be removed through proteolysis