Central Dogma: DNA Replication

• The central dogma of molecular biology describes the flow of genetic information: DNA to RNA to Protein.
• This flow includes DNA replication, transcription, and translation.

DNA Replication

• DNA Replication is semi-conservative, meaning each new molecule contains one parent template strand and one new daughter strand.
• Initiation is the first step.
• Helicase unwinds DNA, forming a replication fork.
• Multiple replication forks along a DNA molecule create replication bubbles.
• Elongation is the second step, where new nucleotides are added.
• RNA Primase adds a complimentary RNA primer to each template strand as a starting point for replication.
• DNA Polymerase reads the template strand (3' to 5') and adds new complimentary nucleotides (5' to 3').
• DNA synthesized in the direction of the replication fork is called the leading strand.
• DNA polymerase can only add new nucleotides in the 5' to 3' direction.
• Replication occurs in two directions due to the antiparallel nature of DNA.
• The lagging strand involves RNA primers laid down, with new nucleotides added 5' to 3' moving away from the replication fork.
• The segment of DNA produced on the lagging strand is called an Okazaki fragment.
• The leading strand is synthesized continuously as DNA unwinds and DNA polymerase adds nucleotides.
• The lagging strand is synthesized discontinuously, requiring new RNA primers and Okazaki fragments.
• Termination is the third step.
• A different type of DNA polymerase removes the RNA primer and replaces it with DNA.
• DNA ligase joins Okazaki fragments with phosphodiester bonds to produce a continuous chain.
• Each new DNA molecule is rewound by helicase, and each molecule is identical.
• The leading strand uses 1 primer and is synthesized 5' to 3' continuously.
• The lagging strand uses multiple primers and is synthesized 5' to 3' discontinuously.
• In humans, DNA polymerase adds 50 nucleotides/second.
• DNA polymerase can proofread its own work and does excision repair.
• There is approximately 1 error in 10,000 bases.
• After proofreading, the rate of mutation is 1 in 10,000,000.

RNA Synthesis: Transcription

• RNA is an important type of nucleic acid involved in protein production.
• RNA carries the instructions of DNA out of the nucleus to the ribosomes.
• The genome contains information encoded in genes, transcribed into 4 types of RNA.
• mRNA (messenger RNA) encodes the amino acid sequence of a polypeptide.
• tRNA (transfer RNA) brings amino acids to ribosomes during translation.
• rRNA (ribosomal RNA), along with ribosomal proteins, makes up the ribosomes.
• snRNA (small nuclear RNA), with proteins, forms complexes for RNA processing in eukaryotes.
• Messenger RNA carries the code that specifies the amino acid sequence in a polypeptide.
• Making mRNA starts with a protein encoding gene on a template strand of DNA.
• Initiation is the first step and involves RNA polymerase binding to a promoter region on DNA.
• RNA Polymerase binds to a promoter which is a region of bases that signals the beginning of a gene
• RNA Polymerase is bound to the TATA box of the promoter by transcription factors
• The double helix unwinds and is ready to be transcribed.
• Elongation is the second step.
• RNA Polymerase moves along the protein encoding gene, adding new RNA nucleotides (5' to 3') complimentary to the DNA template.
• Elongation works at up to 60 nucleotides/second.
• Termination is the third step.
• RNA Polymerase reaches the terminator region of the protein encoding gene, releasing all enzymes and factors.
• The product of these 3 steps is called immature or pre-mRNA.

RNA Processing

• Most eukaryotic protein encoding genes contain non-coding segments called introns, breaking up the amino acid coding sequence into segments called exons.
• RNA Processing includes modification and splicing.
• Modification involves adding a modified GTP cap at the 5' end, which serves as a recognition signal for ribosomes.
• A poly(A) tail of 150 or more adenine nucleotides is added to the 3' end, playing a role in mRNA stability.
• Splicing (intron removal) involves snRNPs binding to form the spliceosome.
• The intron loops out, is excised, and exons are spliced together, resulting in mature mRNA.

Protein Synthesis: Translation

• The language of nucleic acids is translated into the language of proteins.
• Nucleic acids have a 4-letter language, while proteins have a 20-letter language.
• If 3 RNA bases code for 1 amino acid, RNA could code for 4^3 = 64 amino acids.
• More than enough coding capacity for 20 amino acids.
• Code is redundant for most amino acids
• The "players" in protein synthesis include mRNA, ribosomes, tRNA, and amino acids.
• Messenger RNA (mRNA) is synthesized in transcription and composed of codons.
• Codons are 3-base sequences of mRNA.
• Ribosomes are made of rRNA and protein, with 2 subunits that form a 3D groove.
• The two major sites on ribosomes are P site (holds the growing polypeptide) and A site (where new amino acids enter).
• Transfer RNA (tRNA) carries amino acids to the ribosome.
• During tRNA charging each tRNA picks up an amino acid from the INP.
• tRNA has a 3-base anticodon that pairs with the mRNA codon.
• There are 20 amino acids, each with a basic structure, held together by peptide bonds.
• Translation has 3 steps: Initiation, Elongation, and Termination.
• Initiation involves the 5' G-cap of mRNA binding to the ribosome.
• The start codon AUG and anticodon with Methionine bind the P site, and the A site is ready to receive new tRNAs.
• Elongation involves adding new amino acids.
• Codon recognition occurs, followed by peptide bond formation.
• Translocation is the movement of the ribosome along the mRNA, with aminoacyl tRNA shifting from the A site to the P site.
• Termination occurs when a stop codon is reached (UAA, UAG, UGA) and all parts release.

Translation, Polypeptides, and Mutations

• Normally, translation of the genetic code produces the correct protein from a long chain of amino acids.
• Translation of codons is dependent on the reading frame, which is a grouping of codons.
• Mutations are any change in the nucleotide sequence of DNA.
• Mutations can involve large sections of chromosomes or single base pairs and can change the reading frame of a gene transcript.
• Changes in one or a few bases is called a Point Mutation
• Two Types of mutations: Substitution or Insertion/Deletions
• Deletion or insertion mutations are most disruptive because they change the reading frame, causing a frame shift.
• Substitution mutations have varied impact on amino acid sequences.
• Substitutions of the 1st or 2nd base in a codon almost always changes the amino acid.
• Substitution of the 3rd base in a codon does not always change the amino acid.
• Mutations are caused by errors in DNA Replication, errors in chromosome crossover in meiosis, or mutagens.
• Mutagens are physical or chemical factors that cause mutations like UV radiation, X-Rays, and chemicals like DDT.
• Many mutations are harmful to the organism.
• Some mutations are beneficial and help the organism to survive.
• If mutations are present in gametes, they can be passed on to offspring, which is the driving force of Natural Selection.