Bio 340: Module 2 - Transcription, Translation, and Mutations

Transcription & Translation and Mutations

Overview

  • Transcription and translation are the two processes through which genes are made into proteins.
  • Key topics include:
    • Transcription and translation mechanisms
    • mRNA and protein sequence prediction
    • Regulation of gene expression
    • Origins and effects of mutations
    • Methods for studying gene expression

Transcription and Translation

  • Transcription: The information in DNA is converted into information carried by RNA.
  • Translation: The information in mRNA is converted into information carried by a protein.
  • Transcription and translation are how DNA is made into proteins.
  • Proteins perform essential functions in the body.

DNA Replication vs. Transcription vs. Translation

  • DNA replication: The process of making copies of DNA during mitosis & meiosis.

Transcription and Translation Timing

  • Transcription and translation occur constantly.
  • mRNA and proteins have short half-lives.
    • mRNA: approximately 10 hours (if you start with 100 mRNA molecules, only 50 will remain after 10 hours).
    • Protein: approximately 40 hours.
  • Cells must continuously produce mRNA and proteins.

Importance of Proteins

  • Proteins perform all the functions in the human body.
  • Genes for traits like flower color are useless unless they are expressed as proteins.
  • Proteins help make pigments, which determine flower color.

Parts of a Gene

  • Genes have two types of parts:
    • Parts that determine when and where the gene is turned on (promoter, enhancer, silencer).
    • Parts that determine protein sequence (exons).

Steps in Transcription

  1. Transcription factors bind to DNA at the enhancer & silencers
  2. RNA polymerase binds to the promoter
  3. RNA polymerase starts transcription
  4. Premature mRNA becomes mature mRNA
1. Transcription Factors Bind DNA
  • Transcription factors are proteins that bind to specific DNA sequences in a gene and regulate gene expression.
  • Activator transcription factors bind to a gene’s enhancers and increase the expression of the gene.
  • Repressor transcription factors bind to a gene’s silencers and decrease the expression of the gene.
  • To start transcription:
    • Activators bind to enhancers.
    • Repressors fall off the silencers.
2. RNA Polymerase Binds to the Promoter
  • Transcription factors open up the DNA and stabilize the RNA polymerase, allowing it to bind.
  • RNA polymerase is a large protein.
  • RNA polymerase makes RNA from DNA.
  • Once it binds, transcription starts!
3. RNA Polymerase Starts Transcription
  • RNA polymerase reads the template strand (from 3’ to 5’ direction) to create a premature mRNA molecule.
  • RNA polymerase reads through untranslated regions AND exons AND introns.
  • The process stops when RNA polymerase hits a stop sequence.
  • The stop sequence is AFTER the stop codon.
4. Premature mRNA Becomes Mature mRNA
  • Premature mRNA contains introns, exons, and untranslated regions.
  • Introns are removed, and a poly-A cap is added to create mature mRNA.
  • Mature mRNA has exons and untranslated regions only.
  • Mature mRNA moves into the cytoplasm for translation.

Final Product of Transcription

  • The final product is a mature mRNA molecule.
  • Mature mRNA consists of untranslated regions & exons of a gene.
  • Untranslated regions regulate when and where translation occurs.
  • Untranslated regions are transcribed but NOT translated.
  • The mature mRNA molecule contains the information to make a protein.

How Transcription Occurs

  • RNA is made using DNA as a template, so DNA encodes mRNA.

Steps in Translation

  1. Ribosome binds mRNA at AUG (start codon).
  2. tRNAs carrying amino acids match to the codons in mRNA.
  3. Polypeptide chain elongates.
  4. Ribosome hits stop codon; release factor comes in & ends translation.
  5. Polypeptide chain folds into a protein.
1. Ribosome Binds mRNA at AUG (Start Codon)
  • The ribosome is a protein-RNA complex that makes proteins from mRNA.
  • The ribosome has small and large subunits.
  • First, the small subunit of the ribosome binds.
  • Then, the tRNA that matches to the start codon binds.
  • Then, the large subunit of the ribosome binds, and translation begins.
2. tRNAs Carrying Amino Acids Match to the mRNA
  • tRNAs are transfer RNAs that carry a specific amino acid.
  • The amino acid matches the anticodon sequence of the tRNA.
  • The anticodon sequence is 3 nucleotides long and matches the codon in mRNA.
3. Polypeptide Chain Elongates
  • The ribosome keeps moving down the mRNA.
  • More tRNAs come in, forming the polypeptide chain.
  • A polypeptide chain is a string of amino acids.
4. Ribosome Hits Stop Codon; Release Factor Comes In & Ends Translation
  • When the ribosome hits one of the three stop codons, the release factor binds to stop codons.
  • The ribosome falls off, and translation ends.
  • There is no “stop” amino acid; translation simply stops.
5. Polypeptide Chain Folds into a Protein
  • The polypeptide chain folds up to become a protein.
  • The 3D shape of the protein determines its function.

Predicting mRNA and Protein Sequence

  1. Determine which DNA strand is coding vs. template (look for start codon!).
  2. Figure out the mRNA sequence.
  3. Figure out what the codons are.
  4. Translate!
1. Identifying Coding vs. Template Strand
  • RNA polymerase needs to determine the coding vs. template strand.
  • Coding strand: Identical to the mRNA, except it has thymines (Ts) instead of uracils (Us).
  • Template strand: The strand that RNA polymerase reads to make the mRNA.
  • The coding strand should have an ATG (start codon) in the 5’ – 3’ direction.
2. Determining the mRNA Sequence
  • Take the coding strand and replace all thymines (T) with uracils (U) to get the mRNA sequence.
3. Identifying Codons
  • Codons are sets of three nucleotides that correspond to an amino acid.
  • The start codon determines the first set of codons.
  • The start codon ensures the correct reading frame is chosen.
  • There are three possible reading frames, but only one is correct.
4. Translation
  • Start at the start codon and continue until you hit a stop codon.
  • There is no STOP amino acid; it just stops.
  • The start of a protein is the N-terminus (N), and the end is the C-terminus (C).
  • Match each codon with its corresponding amino acid.

Why Aren’t All Genes “On” All the Time?

  • Genes are turned “on” or “off” depending on the internal and external environment.
  • Mutations can also affect whether a gene is “on” or “off.”
  • Every cell in the body has the entire genome.
  • Different cells differ in which genes are turned on or off.
  • Sperm and egg cells have only half of the genome.

Gene Expression

  • Gene expression: When a gene is turned “on” (being made into mRNA and protein).
  • Gene expression changes based on:
    1. Stage of development
    2. Internal environment
    3. External environment
    4. Mutations in the gene
Stage of Development
  • During development, different genes are turned on to form legs, arms, brains, etc.
  • Turning on the correct genes at the right time is necessary for proper development.
Internal Environment
  • Multicellular organisms have different tissues that serve different functions and require different proteins.
  • Different tissues express different genes (e.g., brain cells express 8k of 25k genes).
  • Pregnancy alters a person's internal environment, changing gene expression to support the growing fetus.
    • Increase in expression in genes involved in oxygen transport since the fetus needs oxygen.
External Environment
  • Environmental changes (season, sickness, sun exposure, stress, diet) affect gene expression.

Gene Expression Levels

  • Genes aren’t just on or off; they can be expressed at low vs. high levels.

Gene Regulation

  • Gene regulation: How the cell controls what genes are expressed.
  • Up-regulated: Increase in gene expression.
  • Down-regulated: Decrease in gene expression.
  • Gene regulation control can happen during transcription.
  • Transcription factors regulate gene expression.
Transcription Factors and Transcription
  • Transcription factors can attract or block RNA polymerase.
  • They can methylate or un-methylate DNA.
    • Methylating DNA is completely reversible.
    • Less methylated DNA makes it easier for RNA polymerase to bind.
  • They can modify histones to open or close chromatin.
    • More open chromatin makes it easier for RNA polymerase to bind.
Gene Regulation as a Combinatorial Process
  • Any one gene has tens or hundreds of transcription factors that bind to its enhancers and silencers to determine if the gene is off or on
Origin of Transcription Factors
  • Growth factors (hormones) bind to receptors, activating transcription factors, which then up-regulate genes involved in division and growth.

Mutations

  • Mutations: A change in a DNA sequence that creates genetic variation.
  • Mutations create alleles, which are different versions of the same gene.
  • Alleles can affect how the gene and its protein work.

Sources of Mutations

  1. Errors in DNA replication: Mistakes during DNA copying in mitosis or meiosis.
  2. The environment: Radiation, sunlight, chemical mutagens, some viruses.
  3. Spontaneous lesions: Naturally occurring damage to DNA that leads to nucleotide changes.

DNA Repair Mechanisms

  • Our bodies have many mechanisms for finding and fixing mutations.

Mutation Inheritance

  • Parents pass on 10 – 100 new mutations to their children.
  • Older dads pass on more mutations; children born to older dads are more likely to get autism.

Classifying Mutations

  • Mutations can be classified by:
    • Somatic vs. germ-cell
    • Phenotypic effect
    • Type of DNA sequence change
Somatic vs. Germ-Cell Mutations
  • Somatic mutations: Occur in body cells, cannot be passed down, and can affect the phenotype of body cells.
  • Germ-cell mutations: Occur in cells that make sperm and eggs and can be passed onto the next generation.
Phenotypic Effect
  • Most mutations do not affect phenotype because they occur in non-functional parts of the genome or are silent mutations.
  • Mutations can affect phenotypes by:
    • Changing the structure of a protein (mutations in exons).
    • Changing gene expression (mutations in introns, promoters, enhancers, silencers).
DNA Sequence Changes
  • Common types of DNA sequence changes:
    • Insertion: Addition of nucleotides.
    • Substitution: Replacement of one nucleotide with another.
    • Deletion: Removal of nucleotides.
    • Insertions and deletions in exons can cause frameshift mutations.

Methods for Studying Gene Expression

  • RNAseq.
  • Reporter genes.
RNAseq
  • RNAseq lets you see how much every gene in the genome is being expressed
Reporter gene
  • Reporter gene lets you see where in the organism the gene is expressed.
  • Attach regulatory regions (enhancers, silencers, promoters) to a glowing protein (e.g., green fluorescent protein (gfp)).

Key Terms

  • transcription
  • translation
  • RNA polymerase
  • mRNA
  • transcription factors
  • activator transcription factors
  • repressor transcription factors
  • ribosome
  • tRNA
  • coding vs. template strand
  • codons
  • Anticodons
  • Polypeptide chain
  • gene expression
  • gene regulation
  • up-regulation
  • down-regulation
  • mutation
  • DNA replication
  • spontaneous lesions
  • DNA repair
  • somatic vs. germ cell mutation
  • insertion
  • substitution
  • deletion
  • RNAseq
  • reporter genes