Unit 6: Gene Expression and Regulation

🧬 DNA Structure

• DNA is made of repeated subunits of nucleotides

  • Each nucleotide has a 5-carbon sugar, a phosphate, and a nitrogenous base

  • Sugar phosphate backbone of DNA - nucleotides are linked together by phosphodiester bonds between their sugars and phosphates

    • Serves as a scaffold for the bases

• The two DNA strands run in opposite directions

  • “antiparallel“

  • 5’ end or 3’ end depends on which carbon of the sugar ends that strand

  • the 5’ end has a phosphate group at the end

  • the 3’ end has a hydroxyl group at the end (OH)

• Adenine and Thymine bind with 2 hydrogen bonds

• Guanine and Cytosine bind with 3 hydrogen bonds

• DNA is wrapped around proteins called histomes, and the histomes are bunched together in groups called a nucleosome

• When the genetic material is in a loose form in the nucleus, it is called euchromatin, and all of its genes are active and available for transcription

• Prokaryotes have one circular chromosome, and eukaryotes have linear chromosomes

• Plasmids - circular DNA sections that replicate independently of chromosomes

🧬🧬 DNA Replication

• One DNA Strand is called the leading strand and is made continuously as it goes in the 5’ to 3’ direction because nucleotides can only be added to the 3’ end of the growing chain

  • However, DNA polymerase READS the TEMPLATE DNA strand from 3’ to 5’, synthesizing the complementary strand from 5’ to 3’

• The other strand is called the lagging strand and must be built in parts called Okazaki fragments because DNA polymerase doesn’t work in the 3’ to 5’ direction of this strand

  • Okazaki fragments will be joined together by ligase later

• A few bases at the very end of a DNA molecule cannot be replicated because DNA polymerase needs space to bind template DNA

  • Every time replication occurs, the chromosome loses a few base pairs

  • The genome has compensated for this by putting less important DNA at the ends of the molecule called telomeres

    • Telomeres are repeated sequences of nucleotides that are unimportant in terms of genetic information

    • Hayflick limit - the number of times a normal somatic, differentiated human cell population will divide before cell division stops

      • built-in mechanism that prevents cells from undergoing oncogenic transformation

    • Quantative PCR or Sourthern-Blot analysis can be used to measure telomere length

Enzymes

• Helicase unwinds the double helix into two strands

• DNA polymerase adds nucleotides to an existing strand

  • Catalyzes the reaction to pair nucleotides on the template strand with complementary bases on the new strand

• Ligase brings together the Okazaki fragments

• Topoisomerase cuts and regions the helix

• RNA primase catalyzes the synthesis of RNA primers to initiate DNA replication, providing a starting point for DNA polymerase to begin adding DNA nucleotides

🔗 Central dogma of biology

• DNA gets turned into RNA (transcription)

  • Takes place in the nucleus for Eukaryotic cells

• RNA is then sent out into the cell and gets turned into protein (translation)

  • Takes place in the cytoplasm for Eukaryotic cells

  • Both Transcription and Translation take place in the cytoplasm for Prokaryotes

• Most expressed DNA turns into proteins

• Proteins regulate almost everything that occurs in the cell

• DNA → RNA → Proteins

🧩 RNA Structure

• Differences:

  • Single stranded unlike DNA

  • The 5 carbon sugar in RNA is ribose, not deoxyribose like DNA

  • Uracil replaces thymine as adenine’s partner

  • DNA is passed from generation to generation, while RNA is only there for a short time use

• 3 main types of RNA:

  • mRNA (messenger RNA) - temporary RNA version of a DNA recipe that gets sent to the ribosome

  • rRNA (ribosomal RNA) - produced in the nucleolus, makes up part of the ribosomes (the sites of protein synthesis)

  • tRNA (transfer RNA) - shuttles amino acids to the ribosomes. Responsible for bringing the appropriate amino acids into place at the appropriate time by matching anticodons to codons

    • Does this by reading the message carried by the mRNA

  • There is also another type - RNAi (interfering RNA)

    • Small snippets of RNA that can bind to specific sequences of RNA and mark them for destruction

📜 Transcription

• makes an RNA copy of the DNA code

• Steps of transcription are similar to DNA replication

  • One major difference is that transcription only needs to copy the bit of DNA that needs to be expressed

  • Prokaryotes transcribe a recipe that can be used to make several proteins (polycistronic transcript)

  • Eukaryotes tend to have one gene that gets transcribed to one mRNA and translated into one protein (monocistronic)

• Three phases of transcription

  • initiation - helicase unzips DNA

    • transcription begins at special sequences of the DNA called promoters

    • we only need to copy one strand of DNA because RNA is single-stranded

      • the strand we use as a template for RNA is called the antisense/noncoding/minus/template strand

      • the strand that remains dormant is the sense/coding strand

  • elongation

  • termination

• RNA polymerase build RNA from 5’ to 3’ just like DNA polymerase

  • however, RNA polymerase doesn’t need a primer

• Once RNA polymerase finishes adding nucleotides and reaches the termination sequence, it separates from the DNA template which completes transcription

✂ RNA Processing

• In eukaryotes, the RNA must be processed before it can leave the nucleus

• Right now it is called hnRNA right after transcription which contains both coding and noncoding regions

  • Regions that will express the code and be turned into proteins are exons

  • The noncoding regions in mRNA are introns

• Splicing - Introns must be removed before the mRNA leaves the nucleus

  • Done by spliceosome

• A poly(A) tail is added to the 3’ end and a 5’ GTP gap is added to the 5’ end

💪 Translation

• mRNA —> protein

• Translation also involves three phases: initiation, elongation, and termination.

• Process occurs on ribosomes in cytoplasm and on the rough endoplasmic reticulum

• 3 nucleotides is called a codon. Each codon corresponds to a particular amino acid.

• mRNA attaches to the ribosome to initiate translation

• mRNA passes through 3 sites on the ribosome:

  • A site, P site, E site

• mRNA is read in triplets of 3 nucleotides, called codons

  • Each tRNA has a region called the anticodon that is complementary to the codon

  • One end of the tRNA carries an amino acid. The other end, called an anticodon, has three nitrogenous bases that can complementarily base pair with the codon in the mRNA.

  • The third position is said to experience wobble pairing. Things that don’t normally bind will pair up, like guanine and uracil.

• tRNA binds with amino acids and “shuttles“ them to the ribosome

• the amino acid will then be added to the growing polypeptide

🫛 Gene Expression

• regulated primarily by:

  • transcription factors influencing transcription (pre-transcriptional regulation)

  • RNAi after transcription (post-transcriptional regulation)

• regulation is dynamic and can increase or decrease gene expression, RNA levels, and protein levels based on the cell’s needs

🔄 Mutations

• mutations can result from changes in the DNA message or mRNA message

• can be small (single nucleotide swaps, additions, or deletions) or large (big chunks or entire chromosomes are swapped, duplicated, or deleted)

🧪 Biotechnology

• recombinant DNA

• polymerase chain reaction (PCR)

  • method used to make millions to billions of copies of a specific DNA sample rapidly

  • allows scientists to amplify a very small sample of DNA sufficiently to enable detailed study

• Quantitative PCR

  • a technique that amplifies and quantifies a target DNA sequence during the reaction

  • It is used to determine the quantity of DNA or RNA in a sample

• transformation of bacteria

  • phage infecting bacteria

  • involves changing the bacteria’s DNA

• gel electrophoresis

  • see last unit

  • The DNA moves because of the negatively charged Sugar-Phosphate backbone

🦠 Pathogens

• bacteria and viruses or common pathogens

• bacteriophages are viruses that infect bacteria

• viruses require a host to replicate and sometimes lyse (disintegrate the cell membrane) the host cell during infection