Unit 3 - Molecular Genetics Flashcards
Griffith’s experiment
In 1928, pneumonia was a deadly virus at the time, and Frederick Griffith was studying creating a vaccine. He injected mice with two strains of it; a smooth (S-) strain, and a rough (R-) strain. The S-strain was virulent and killed the mouse, but the R-strain was non-virulent, and when injected, the mouse still lived. Griffith also recognized that he could heat-kill the R-strain; rendering it non-virulent. When a mouth was injected with the S-strain and the heat-killed R-strain, however, the mouse still died. Griffith theorized that the heat-killed virulent bacteria of the R-strain passed on its traits to the living non-virulent bacteria of the S-strain; rendering it deadly. He labelled this, the “transforming principle” and it suggested that bacteria were capable of transferring genetic information.
Hershey-Chase experiment
Alfred Hershey and Martha Chase were a microbiologist team in 1952 who were determined to figure out whether DNA or proteins contained genetic code. They used bacteriophages; viruses that infect bacteria - specifically the T2 bacteriophage strain of a virus, which contains a protein coat that surrounds DNA. To study the role protein and DNA alike play in T2 infection, Hershey and Chase used radioisotopes to trace each molecule. They used a radioisotope of sulphur to trace for proteins (as proteins contain sulphur, but DNA doesn’t), and they likewise used a radioisotope of phosphorus to trace for DNA (as DNA contains phosphorus, but proteins don’t). The bacteriophages (both phosphorus-labelled and sulphur-labelled) injected E. coli bacteria, which were then agitated with a blender and centrifuged, and when the infected bacterial cells and the liquid phage “ghosts” were separated, Hershey and Chase found that most of the radioactivity in the phosphorus-labelled phage solution was found in the bacterial cells, but most of the radioactivity in the sulphur-lablelled phage solution was found in the phage shell medium. With this data, the two scientists concluded that the viral DNA was transferred into the bacterial cells, and was therefore was contained the genetic information.
DNA
- hereditary molecule that passes traits on to the next generation
- contains information (nucleotide sequences) that codes for proteins (genes)
- polymer composed of nucleotides
- hydrogen bonds hold complementary bases together in double-stranded DNA
genome structure
- diameter = 2 nm
- 1 base-pair = 0.34 nm
- 1 revolution = 10 base-pairs
- 1 revolution = 3.4 nm
nucleotide
- composed of a phosphate group, pentose sugar, and nitrogenous base (is a phosphorylated nucleotide)
- sugar-phosphate backbone linked by phosphodiester bonds
- pentoses and nitrogenous bases linked by glycosyl bonds
nucleoside
composed of a pentose sugar and nitrogenous base
purines
- adenine and guanine
- composed of 2 rings
pyrimidines
- thymine, cytosine and uracil
- composed of 1 ring
DNA vs. RNA
DNA
- composed of deoxyribose sugars (hydrogen in carbon 2)
- more stable
- used for CSI and archaeology
- found in the nucleus
RNA
- composed of ribose sugars (hydroxyl in carbon 2)
- less stable; degrades much faster over time than DNA
- is an umbrella term; there are many different types, each with their own structures, functions, and locations (mRNA, rRNA, mtRNA, tRNA, iRNA, gRNA, etc.)
The Central Dogma
a dynamic (non-unidirectional) process that states how DNA contains instructions for making a protein:
DNA ⇄ mRNA → proteins
- DNA replication (DNA → DNA)
- DNA transcription (DNA → mRNA) and reverse transcription (DNA ← mRNA)
- DNA translation (mRNA → proteins)
codon
- a triplet of bases
- RNA-based
- results in the transcription of one amino acid
- there are 64 different codons in total (4 bases and 3 positions; 43 = 64)
- start codons (AUG) signal the beginning of protein synthesis
- stop codons (UAA, UAG, UGA) are non-translatable, and simply signal the end of protein synthesis
DNA replication
- a.k.a. natural cloning (in vivo)
- occurs in the nucleus
- takes place at multiple origins of replication - within “replication bubbles” - in eukaryotes, but at only one origin of replication for prokaryotes
- semi-conservative nature of DNA synthesis
- Gyrase/topoisomorase unwinds supercoiled DNA.
- Helicase disrupts hydrogen bonds to make single-stranded DNA via denaturation; forming replication forks.
- Single-stranded binding proteins prevent hybridization.
- RNA primers bind to DNA, via primase, and act as a starting point for DNA polymerase III.
- DNA polymerase III synthesizes DNA, from 5’ to 3’ direction (on the leading strand, this is done continuously; on the lagging strand, this is done in chunks and forms Okazaki fragments between primers).
- DNA polymerase I checks for errors in DNA, and replaces RNA primers with the appropriate nucleotides (uracil to thymine).
- DNA ligase joins remaning gaps together with phosphodiester bonds.
DNA transcription
- occurs in the nucleus for eukaryotes; occurs in cytosol (the nucleoid) for prokaryotes
- coupled with DNA translation in prokaryotes
- Initiation: RNA polymerase binds to a promoter region. (The promoter region is an A=T-rich area, as A=T has two hydrogen bonds, which are easier to break down than the three hydrogen bonds between C≡T. This is therefore energetically favourable for RNA polymerase.)
- RNA polymerase unwinds the double stranded DNA; exposing the template strand (a.k.a. sense strand), and the complimentary coding strand (a.k.a. anti-sense strand).
- Elongation: mRNA is synthesized within the “transcription bubble”, in 5’ to 3’ direction. It has the same sequence as the coding strand, but with uracil instead of thymine.
- As RNA polymerase moves along DNA, mRNA is continuously synthesized, and DNA that has already been synthesized is rewound.
- RNA reaches the end of the gene (the “termination sequation”).
- Termination: RNA synthesis ends; mRNA is released, and DNA and RNA polymerase are recycled to form more mRNA.
post-transcriptional modifications
- occur in the nucleus
- added to the primary transcript (mRNA without modifications) to form the full mRNA transcript (mRNA with modifications)
5’ capping
- 5’ cap of modified guanine nucleoside triphosphate added on the 5’ end of mRNA
- protects the primary transcript from being broken down by nucleases and RNAses
poly-A addition
- poly-A polymerase adds a tail of 200-400 A’s to the 3’ end mRNA
- acts as a guidance mechanism to rough endoplasmic reticulum or ribosomes
splicing
- spliceosomes excise introns (non-translatable mRNA regions that stay in the nucleus), and join together exons (translatable mRNA regions that exit the nucleus)
DNA translation
- made possible with ribosomes, mRNA and tRNA
- one mRNA can result in thousands of polyptides
- amplication factor of greater than 106 cells
- coupled with DNA transcription in prokaryotes
- mRNA is pulled through the ribosome; the start codon establishes the reading frame.
- tRNA is brough into the P-site (peptidyl site); the anticodon of the tRNA is complimentary to the codon of the mRNA and carries the amino acid coded by the start codon.
- A second tRNA enters the A-site (aminoacyl site). A peptide bond forms between the two amino acids.
- The ribosome translocates one codon over, and the next tRNA brings in the appropriate amino acid into the A site; the first tRNA exits the ribosome, and is recycled for later use.
- This process is repeated until the ribosome reaches a stop codon, for which no tRNA exits.
- The ribosome-mRNA complex is dismantled, and the polypeptide chain is released.
