protein synthesis/genetic mutations/ genetic engineering

proteins: macromolecules

building block: amino acids (bonded w polypeptide bonds)
- coded by a sequence of 3 nucleotides (codons)
ex. transport/membrane proteins, enzymes, muscle tissue

genetic code: DNA sequence that gives instructions to make proteins

RNA: ribonucleic acid

single stranded
nucleotide w ribose sugar, phosphate, and 4 bases (a, u, c, g)
found in nucleus, cytoplasm (ribosomes)
3 types
- mRNA (messenger)
- takes info from DNA in the nucleus and brings it to ribosome
- found in nucleus, cytoplasm, ribosome
- tRNA (transport)
- takes amino acids to ribosomes and assembles into proteins
- found in cytoplasm, ribosome
- rRNA (ribosomal)
- loading dock for mRNA/tRNA
- found in ribosome (makes up ribosomes?)

scientific dogma: an established principle believed by scientists

central dogma of biology: information is transferred from DNA → RNA → proteins (all DNA codes for proteins)

protein synthesis

2 parts
- transcription: making mRNA using DNA as a template → like DNA replication but simpler
- DNA = recipe book
- translation: taking RNA and changing it to proteins
codons: set of 3 mRNA bases that code for an amino acid
- 64 possible codons
anticodons: set of 3 tRNA bases (carry amino acid and match up w mRNA codons [complementary bases])

part 1: transcription ==[diagram]==

occurs in the nucleus (eukaryotes) and nucleoid region (prokaryotes)
makes mRNA as a messenger for DNA’s genetic info/instructions as it never leaves the nucleus
RNA polymerase does all jobs: unzips/unwinds, adds new nucleotides, etc.
- builds on leading strand from 5’ to 3’
~~steps~~
- ~~DNA unzips~~
- ~~mRNA nucleotides pair up with 1 DNA strand only (leading strand)~~
- ~~complementary base pairs ( A → U, T → A, C → G, G → C)~~
- ~~when completed copying DNA information, mRNA floats off and travels to ribosome / DNA strands come back together (temporarily split to be used as template)~~
3 stages
- initiation: start transcription as RNA polymerase binds to a promoter region and starts to unwind DNA and add RNA bases
- elongation: RNA polymerase continues to add complementary bases
- termination: ends transcription as RNA polymerase lands at the stop/termination region and mRNA molecule floats off

post transcriptional modification

DNA has different sections of nucleotides: “junk” introns that don’t code for proteins and “good” exons that do
before leaving the nucleus, the introns are spliced out of the mRNA sequence of the exons are put together

part 2: translation ==[diagram]==

occurs at the ribosome
information carried by mRNA assembles amino acids carried by tRNA to make proteins (amino acid chain/polypeptide)
- start codon: starts translation (green light / capital letter) → AUG (met)
- stop codon: ends translation (red light / period) → UGA, UAA, UAG
ribosome ==[diagram]==
- A: attachment site (for tRNA)
- P: peptide bond site where peptide bonds form
- E: exit site (tRNA leaves ribosome after unloading amino acid)
steps
- mRNA attaches to ribosome at start codon (travels to ribosome through nuclear membrane and cytoplasm w microtubules)
- start codon (AUG) matches with tRNA anticodon (with amino acid methionine) at P site
- complementary bases
- next codon matches with tRNA anticodon (with an amino acid) at A site
- the 2 amino acids join together w peptide bonds
- amino acid at P “jumps ship” to A site
- tRNA at P moves to E and exits the ribosome after unloading its amino acid
  - can be reused → goes to find new amino acid
- tRNA at A moves to P with attached amino acids
- new tRNA comes at A site and attaches more amino acids to growing chain w ^
- repeats until ribosome reaches a stop codon → protein is completed and floats away to be used

codon chart → use to find what amino acid is coded by a certain codon/DNA sequence/etc.

if an mRNA codon chart → reference mRNA codon sequence to find amino acid
if mRNA codon is AUG → find A on left side, U on top side, G on right side

base pairing practice

DNA: TAT / GGA / CAG / TAG
mRNA: AUA / CCU / GUC / AUC
tRNA: UAU / GGA / CAG / UAG
amino acids: ile / pro / val / ile

~~cell organelles~~

~~ribosomes (free): make proteins to be used by the cell~~
~~ribosomes on rough ER: make proteins to be shipped out of the cell~~
- ~~golgi apparatus: quality check proteins that are being shipping out~~
- ~~proteins shipped out in vacuoles/vesicles~~

DNA replication	both	transcription	both	translation
DNA polymerase, helicase, primase, ligase	polymerase enzyme	RNA polymerase	uses mRNA	uses tRNA and rRNA
makes DNA (double stranded)	DNA is a template	makes mRNA (single stranded)	part of protein synthesis	makes a protein
both strands used as template	nucleotides added 5’ → 3’ with complementary base pairs	one strand used as template	base pairs are A, U, C, G	in ribosomes
base pairs are A, T, C, G	in the nucleus		occurs in G1/G2
occurs in S phase prior to cell division

genetic mutations

genes: controls what an organism looks like / its inherited traits by controlling protein shape and structure
everyone has distinct DNA/genomes
- polymorphisms = variations in DNA/genes
- account for slight differences in people ex. hair color / but can also cause disease
- although all polymorphisms are the result of a gene mutation, only polymorphisms that are not within “normal variations” are called mutations
how mutations occur → everyone accumulates changes in DNA throughout lives
- copying errors in DNA replication
- DNA damage through environmental agents ex. radiation, chemical mutagens
- inherited/hereditary
- mutations occurring in somatic (body) cells, ex. skin cells, are not hereditary
- germ line mutations are mutations in gametes (egg/sperm) and are hereditary / passed down from parent → child
  - responsible for hereditary diseases
DNA repair of mutations does not work as effectively as we age, causing accumulation of mutations and aging
types of mutations
- any change in a gene sequence of bases can alter the gene’s meaning and change the protein that is made, or how or when a cell makes the protein
- 2 large classes: point mutation and frame shift mutation
- point mutation → substitution
  - change/replacement in one or more bases of a gene sequence (none are added/deleted)
- frame shift mutation → insertion or deletions
  - one or more bases are inserted or deleted → because cells read DNA in three-base codons, this changes each subsequent codon / “shifts”
- inversions
  - a section of DNA is reversed
- DNA expression mutation
  - do not change the protein but where and how much of a protein is made
  - proteins may be made at the wrong time, wrong cell type, or wrong amount
- translocation
  - DNA is transferred to another chromosome
  - reciprocal (even exchange between chromosomes where there is no change in amount)
  - non-reciprocal (one-way transfer resulting in change in amount)
- silent mutation: DNA base change occurs but does not change resulting amino acids
- nonsense mutation: DNA base change occurs and stop codon appears too early, stopping translation too early and resulting in shortened protein
- missense mutation: DNA base change occurs and replaces one amino acid with another, resulting in protein not formed properly

genetic engineering (synthetic biology): making changes in genome (DNA sequence) of organisms

gel electrophoresis ==[diagram]==

agarose solution poured into glass plate casting tray, wells formed with comb
DNA segments fragmented by restriction enzymes (ex. eco r1) are loaded into wells with a micropipette
gel plate is immersed in a charged butter solution → power supply charges side with wells negatively and side far from the wells positively
- negatively charged DNA fragments move through the filter-like gel towards the positive side due to attraction
this separates DNA fragments by length as smaller fragments move faster through holes in the gel
result: DNA fingerprint ==[diagram]==
- unique pattern of bands to each organism as no one has identical DNA (except identical twins)
- length of DNA fragments are smaller as they are farther from the wells → measured by bp (base pairs)
- standard sample is used as a reference for size of samples
purpose: identifying crime suspects, medical (transplants, etc.), parentage
- crime: a suspect’s DNA and the DNA of the perpetrator found at a crime scene would be an exact match
- parentage: all of the baby’s DNA must be a match to either the mom or dad’s DNA in a DNA fingerprint ==[diagram]==

PCR (polymerase chain reaction)

purpose: to make copies of specific DNA fragments
process: based on DNA replication
- denaturation (95 C): separate DNA strands by breaking hydrogen bonds at high temperature
- annealing (55 C): primers added to complementary sequences (“joining of bases”)
- extension (72 C): strand is grown as DNA polymerase attaches complementary bases
enzyme used: taq polymerase → from archaebacteria thermophiles so it has high optimal temperature
2^n = amount of strands produced (n is number of PCR cycles) ==[diagram]==

genetic transformation: changing an organism’s DNA by transferring foreign DNA into host genome

transgenic organism: an organism containing DNA from another species
recombinant: DNA made from combining DNA from two different species
==[diagram]== example: inserting HGH (human growth hormone) into bacterial plasmid to make transgenic bateria
- section of human DNA (HGH gene) cut out by restriction enzyme
- same section cut out from bacterial plasmid by restriction enzyme
- plasmid and cutout HGH gene are bonded with ligase (spliced together) and inserted into bacterial cell
- human protein (HGH) is produced by bacteria
purpose: make proteins for humans/animals (ex. insulin, HGH) more efficiently, sustainably, and ethically
enzymes: ligase, restriction enzyme (ex. eco r1)

gene therapy: fixing DNA by replacing defective gene with normal ones

CRISPR + CAS9 searches for and replaces bad genes with good genes
- purpose: gene therapy / process: genetic engineering
- real life examples: treating diseases like sickle cell

cloning: making genetically identical organisms

process
- nucleus of an egg (haploid) is removed and destroyed of organism A
- empty egg is fused with a somatic/body cell of organism B
- the egg cell, with the nucleus of the body cell, starts dividing
- embryo is placed in the uterus of a foster mother where it develops to full term
- has DNA of organism B
dolly the sheep - cloned by ian wilmut
asexual process for plants