EK

Lecture 8 Notes: DNA to Protein — Transcription, Translation, and Mutations (Vocabulary)

DNA and RNA as languages for polypeptides

  • Gene: a region of DNA that can be expressed to produce a functional product that is either a polypeptide or an RNA molecule.

  • DNA genotype is expressed as proteins: DNA dictates traits via protein synthesis; the molecular 'chain of command':

    • DNA in the nucleus → RNA, then RNA in the cytoplasm → protein

    • Transcription: synthesis of RNA under the direction of DNA

    • Translation: synthesis of proteins under the direction of RNA

  • The central idea: DNA -> RNA -> Protein; mRNA conveys genetic messages from DNA to the translation machinery of the cell.

Central dogma and coding language

  • DNA and RNA use a common nucleotide language to encode proteins; conversion steps include:

    • Transcription rewrites the DNA code into RNA using the same nucleotide language

    • Translation converts the RNA language into an amino acid sequence for polypeptides

  • Codons are the 3-base “words” that specify amino acids; the genetic code is read in triplets and is nonoverlapping.

DNA transcription and mRNA production

  • Transcription produces mRNA from a DNA template strand under the direction of DNA.

  • Strand to be transcribed (DNA template): the strand that is read to synthesize RNA; the RNA produced is complementary to this template.

  • Prokaryotes: transcription and translation occur in the same compartment (cytoplasm).

  • Eukaryotes: mRNA must exit the nucleus via nuclear pores to enter the cytoplasm; mRNA undergoes processing before export.

  • Eukaryotic mRNA features:

    • Introns: interrupting sequences that must be removed

    • Exons: coding regions that will be retained in mature mRNA

mRNA processing in eukaryotes

  • RNA splicing removes introns and joins exons to produce a continuous coding sequence.

  • Exons constituting the mature mRNA are joined and exit the nucleus for translation.

  • Cap and tail additions to mature mRNA:

    • 5′ cap (guanine) protection and ribosome docking

    • 3′ poly-A tail (adenines) protection and export aid

  • Functional roles of the cap and tail:

    • Facilitate export from the nucleus

    • Protect mRNA from enzymatic degradation

    • Help ribosomes recognize and initiate translation

Codons and the genetic code

  • If there are 4 RNA bases and a codon has 3 bases, how many possible codons exist?

    • 4^3 = 64

  • Codon concepts:

    • Triplet code: codon = a three-base unit

    • Translation uses codons to specify amino acids

    • Each amino acid can be specified by one or more codons (redundancy)

  • Translation overview: moving from nucleotide language (RNA) to amino acid language (proteins)

The genetic code: properties

  • Three nucleotides specify one amino acid

  • 61 codons code for amino acids

  • AUG codes for methionine and also signals the start of translation

  • There are 3 stop codons signaling termination of translation

  • Characteristics of the genetic code:

    • Redundant (degenerate): more than one codon codes for some amino acids

    • Unambiguous: any given codon codes for one amino acid (or a stop signal)

    • Nearly universal across organisms

    • No punctuation: codons are adjacent with no gaps

  • Codon base position representation (conceptual): second base, first base, third base interactions determine amino acid specification

Transfer RNA (tRNA) and decoding

  • tRNA molecules = the interpreters that convert mRNA codons into amino acids

  • Roles of tRNA:

    • Pick up the appropriate amino acid and deliver it to the growing polypeptide

    • Use a specific anticodon triplet to recognize the corresponding mRNA codon

  • Key features of tRNA:

    • Anticodon: a triplet in tRNA that pairs with mRNA codon

    • Amino acid attachment site: where the corresponding amino acid is covalently attached

    • Enzyme: aminoacyl-tRNA synthetase attaches the correct amino acid to its tRNA

    • L-shaped folded structure stabilized by hydrogen bonds and modified bases

  • Visual hints from the lecture:

    • tRNA structure includes an amino acid attachment site and an anticodon loop, with chemically modified bases

Ribosomes: the machinery of translation

  • Ribosome = site of translation; coordinates mRNA and tRNA to synthesize protein

  • Composed of two subunits (small and large) containing ribosomal RNA (rRNA) and proteins

  • Subunits assemble on mRNA during translation

  • Ribosome architecture includes:

    • mRNA binding site

    • A site (aminoacyl site) where the incoming tRNA binds

    • P site (peptidyl site) where the growing polypeptide chain is held

    • Exit tunnel through which the polypeptide chain exits

  • Translation dynamics: tRNA carries amino acids to the ribosome; the ribosome catalyzes peptide bond formation between amino acids

Translation in detail: from mRNA to polypeptide

  • Start codon (AUG) marks the initiation of translation and sets the reading frame; encodes methionine

  • Elongation cycle: successive aminoacyl-tRNA delivery to the A site, peptide bond formation, and translocation

  • Stop codons (e.g., UAA, UAG, UGA) terminate translation

  • Summary: mRNA codons are read in triplets; tRNA anticodons pair with codons; ribosome catalyzes peptide bond formation; a polypeptide is synthesized and released

Review questions and conceptual checkpoints

  • Does translation represent DNA -> RNA or RNA -> protein?

    • Translation is RNA -> protein; the information flow is DNA -> RNA -> Protein

  • Where does the information for producing a protein originate: DNA or RNA?

    • DNA provides the template; RNA carries the message to the ribosome

  • Which molecule has a linear sequence of codons: rRNA, mRNA, or tRNA?

    • mRNA has a linear codon sequence; rRNA and tRNA have structural/functional roles with different sequences

  • Which molecule directly influences the phenotype: DNA, RNA, or protein?

    • Protein (the final functional product) most directly influences phenotype; DNA and RNA control protein synthesis

Mutations: changing the meaning of genes

  • Mutation: any change in the nucleotide sequence of DNA

  • Types of mutational changes:

    • Large chromosomal rearrangements

    • Single nucleotide changes (base substitutions)

  • Mutagenesis refers to mutations produced by external factors; mutagens include high-energy radiation and certain chemicals

  • Causes of mutations:

    • Spontaneous errors during DNA replication or recombination

    • Mutagens: X-rays, ultraviolet light, chemicals

Base substitutions and their consequences

  • Base substitutions involve replacing one nucleotide with another

  • Possible outcomes:

    • Silent mutation: no change in amino acid

    • Missense mutation: different amino acid coded

    • Nonsense mutation: creates a premature stop codon, truncating the protein

  • Example motif (illustrative): Normal and mutant hemoglobin sequences show how a single base change can alter the amino acid and protein function

Deletions and insertions: reading frame shifts

  • Deletions or insertions alter the reading frame (triplet grouping) of the mRNA

  • Consequence: codons are read differently, leading to many amino acid changes and often a nonfunctional polypeptide

  • Visual example shows how nucleotide deletions/insertions shift the reading frame from the original sequence

DNA replication, proofreading, and repair

  • DNA replication is not perfect; three major repair mechanisms:

    • Proofreading by DNA polymerase

    • Mismatch repair

    • Excision repair

  • DNA repair dramatically lowers the error rate from roughly 10^{-4} per base to about 10^{-9} per base

  • The repair systems are energetically costly but essential for cell survival

Other sources of DNA damage and cellular responses

  • Sources of DNA damage:

    • Normal metabolism generates reactive oxygen species that can damage DNA

    • Environmental factors: UV radiation, smoking, etc.

    • Mitochondrial DNA damage contributes to overall genomic instability

  • As cells age, DNA repair capabilities decline

  • Cellular responses to unrepaired DNA damage:

    • Dormancy (cell cycle arrest)

    • Programmed cell death (apoptosis)

    • Cancerous transformation if damage accumulates

Extreme examples and real-world relevance

  • Werner syndrome: a human disorder characterized by accelerated aging linked to DNA repair defects

  • Deinococcus radiodurans: a polyextremophile with exceptional resistance to gamma radiation and oxidative damage; studied for potential bioremediation and radiation cleanup

History of DNA:

A man named Friedrich Miescher took some white blood cells and extracted these phosphate rich chemicals from them. he named them nuclein as they came from the ncucleus of the blood cell.

Another man named Frederick Griffith discovered bacterial transformation, where a cell takes in foreign DNA.

  • Through the bacteria that causes pneumonia, he discovered that there are two different types of cultures: the rough (R) and the smooth (S)

  • The rough cultures were non-pathogenic while the smooth cultures were (they have a smooth capsule over them that allows them to escape immune system)

  • How did the experiment turn out? Griffith found that when he had injected a heat killed S strain along with the R strain into the mice, that had killed them. Initially proving that it wasn't just the capsule killing the mice - something had moved from the dead S strain to the R strain, making it effective.

  • Following that study, three other scientists decided to isolate DNA and proteins from the mice and found that when DNA was broken down, it couldn't change the code of the bacteria but when it was active, DNA was the main cause of the transformations. They did this using radioactive phosphate; when the bacteriophage was introduced, the DNA enters the host cell rather than the protein.

AGTC

  • Erwin Chargaff discovered that each of the nucleic acids were in different abundances and they varied from species to species, but his rule that he discovered had to do with the fact that when there is adenine, there is an equal # of thymine and when there is cytosine, there is an equal # of guanine.

  • The total # of pyrimidines (T, C) must always equal the total # of purines (A,C) because of how they pair up.

Nucleotides: The building block of DNA

  • Contains a nitrogenous base at the end of the strand, branched off too a pentose sugar (5 -carbon) and a phosphate group at the other end (the end that doesn't connect)

  • The phosphate makes it acidic, is connected to the 5 carbon group via an ester link.

  • In DNA, there is a hydroxy (OH) group on the third carbon and in RNA, there is a hydroxyl group on both links 2 and 3 of carbon. This makes RNA less stable of a molecule.

  • Nucleotides link together through phosphodiester bonds. The phosphate on the 5′ carbon of one nucleotide connects to the 3′ carbon of the next. This creates a chain that runs in one direction, with a 5′ end (free phosphate) and a 3′ end (free –OH group).

  • A nucleoside is a base and a sugar.

  • A nucleotide is a nucleoside with 1-3 connected phosphate groups.

Watson & Crick:

  • Based on x-rays, these two scientists determined that DNA forms a helix structure

  • A + C bond to form two hydrogen bonds

  • G+C bond to form 3

Sickle cell:

  • Resultant of a mutation of a singular base pair

  • Changes normal hemoglobin codon from "Glu" to "Val"

  • However, having sickle cell gene may be able to protect you from a dangerous disease of malaria.

Other types of mutations (more serious)

  • Insertion: Adding a base, you change reading frame and your codons get screwed up

  • Deletion: Removing a base, and your reading frame gets screwed up

Three types of repair:

  • Proofreading

As cells age:

  • Dormancy

  • Programmed cell death

  • Cancerous states