Chapter 3 - From Genes to Proteins Flashcards
two types of bases?
purines and pyrimidines
types of purines
adenine (A), guanine (G)
types of pyrimidines
cytosine (C), thymine (T), uracil (U)
bases of RNA
adenine, cytosine, guanine, uracil
bases of DNA
adenine, cytosine, guanine, thymine
nucleoside
sugar + base
nucleotide
- a nucleoside with one or more phosphate groups attached
- usually at C5 of the sugar
- can also be at other positions
nucleic acid
polymer of nucleotides
phosphodiester bonds
- links nucleotides by sugar
- creates a nucleic acid in which its sequences are read 5’ to 3’
- these bonds are high in energy because they include phosphate groups that are high in energy
how are two nucleotide strands held together?
- they are held together by hydrogen bonds
- 3 hydrogen bonds form between guanine and cytosine
- 2 hydrogen bonds form between adenine and thymine or adenine and uracil
chargaff’s rule
- the amount of A+G = C+T
- base pairs contain a purine (A, G) and a pyrimidine (C, T, U)
- A-T, A-U, G-C are the possible base pairs
model of DNA
double-stranded, antiparallel, right-handed, diameter of 20 Å, major and minor grooves, backbone exposed to solvent
model of RNA
single-stranded, can fold back on itself, intricate 3D shapes
order of duplex stability from most stable to least stable
RNA-RNA, RNA-DNA, DNA-DNA
X-ray crystallography
- dominant method for deducing high-resolution protein structures
- X-rays scatter as they pass through crystallized protein
- the resulting waves interfere with each other, creating a diffraction pattern from which the position of atoms is deduced
cryo-electron microscopy
- used instead of X-ray crystallography
- beam of electrons is fired at frozen protein solution
- emerging scattered electrons pass through a lens to create a magnified image on the detector, allowing the structure to be deduced
what does the stability of the DNA helix rely upon?
stacking interactions. does not really depend on the hydrogen bonds between base pairs
which base pair is stronger?
G-C
what drives the double helical conformation of DNA?
entropic forces that induce base stacking
different forces that stabilize DNA
- predominant forces: hydrophobicity, base stacking, entropy
- hydrogen bonding in base pairs
- ionic interactions: cations, polyamines
denaturation/renaturation of DNA
- apply high heat to melt DNA and denature it
- cooling the denatured DNA to 20-25 C below Tm will cause renaturation
- sometimes rapid cooling of the denatured DNA to temperatures much lower than Tm leads to improper base pairing
- this improper base pairing can be fixed by rewarming the base pairs to 20-25 C below Tm to cause renaturation
genes
sequences of DNA
replication
process for copying DNA
transcription
process that converts DNA into RNA
reverse transcription
process that covers RNA into DNA. this does not happen in our body
translation
process that makes proteins from RNA template
DNA replication (In vivo)
- DNA must be copied in order to sustain life
- excessive DNA replication can be indicative of cancer
DNA replication (In vitro)
- polymerase chain reaction (PCR) has allowed researchers to study Nucleic Acids and genes
- PCR amplifies DNA
3 types of RNA
messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA)
mRNA
encodes for polypeptide sequences
tRNA
carries amino acids to ribosome
rRNA
aids in polypeptide synthesis
genetic code
- use to translate mRNA into an amino acid sequence
- 64 codons
- codon has 3 nucleotides
- a lot of codons are redundant
how are genes identified?
- scan for open reading frame: start codon (ATG) or stop codon (TAA, TAG, TGA)
- comparison with known genes
what is gene number roughly correlated with?
organismal complexity
relationship between noncoding DNA and the organism
proportion of noncoding DNA generally increases with complexity of the organism
sequence strategy for polymer
1) cleave polymer into small fragments
2) separate fragments
3) determine the sequence of resides in each of the fragments
4) determine the order of fragments in original polymer by overlapping fragments
restriction enzymes
they cut DNA at specific sequences
role of restriction enzymes in nature
- aid in defence for bacteria
- bacteria methylate their own DNA
- bacteriophages (viruses that infect bacteria) have unmethylated DNA
- bacterial restriction enzymes can recognize and excise viral DNA
type 1 restriction enzymes
- activity: endonuclease, methylase
- cleavage site: ≥1000 bp from recognition sequence
type 2 restriction enzymes
- activity: endonuclease
- cleavage site: within or near recognition sequence
- cleave 4-8 bp of dsDNA
- palindromic recognition sequences
- forms blunt or sticky ends
type 3 restriction enzymes
- activity: endonuclease, methylase
- cleavage site: approximately 24-26 bp from recognition sequence
EcoRI
- recognizes 5’-GAATTC-3’
- complimentary sequence is implied
- EcoRI cuts both strands after the 5’-G
- each fragment has sticky ends
EcoRV
- recognizes 5’-GATATC-3’
- complimentary sequence is implied
- EcoRV cuts both strands after the 5’-T
- each fragment has blunt ends
gel electrophoresis
technique used to separate DNA fragments or other macromolecules based on their size or charge. a current is run through the Gell containing molecules of interest, creating a positively-charged side and a negatively-charged side. this allows attraction/repulsion forces to cause the molecule to travel to the opposite end of the cell accordingly
pyrosequencing
- method determines the sequence of residues in each DNA fragment
- steps:
1) DNA polymerase incorporates a complementary nucleotide into the new DNA chain (extends the primer). this releases a inorganic pyrophosphate (PPi).
2) sulfurylase converts the PPi reaction product to ATP
3) luciferase uses the ATP to power a reaction that produces light. the intensity of the light released is used to determine the number of nucleotides incorporated, etc.
illumina sequencing
- used to determine the series of base pairs in the DNA
- steps:
1) DNA polymerase incorporates a complementary nucleotide into the new DNA chain and unreacted nucleotides are washed away
2) the incorporated nucleotide is detected by its fluorescence, then the fluorescent group is removed
3) a new solution of nucleotides is introduced
cloning techniques
1) a fragment of DNA is generated
2) the fragment is incorporated into a vector
3) the vector with the inserted DNA is introduced into cells, where it is replicated
4) cells containing the desired DNA are identified or selected
types of cloning vectors
plasmid, cosmid, bacterial artificial chromosome, yeast artificial chromosome
recombinant DNA
- DNA fragments are joined to produce this
- plasmid, foreign DNA are cut
- then the pieces are annealed in such a way that now the plasmid and foreign DNA are combined into one
- ligation occurs to produce recombinant DNA. this is done by DNA ligase as it facilitates the joining of DNA fragments by catalyzing the formation of the phosphodiester bond
PCR
- polymerase chain reaction
- amplifies DNA to make billions of copies of DNA efficiently and accurately
requirements for PCR
- heat-stable DNA polymerase (ex: Taq DNA polymerase)
- primers (DNA oligonucleotides)
- deoxynucleotide triphosphates (dATP, dGTP, dCTP, dTTP)
- DNA template
- buffer
- thermal cycler: device that heats and cools sample
chemical reactions used and repeated in PCR
1) denaturation:
- dsDNA separates at high temperatures to form ssDNA
- 92-95 C
2) annealing:
- primers can base pair to ssDNA
- approximately 55 C
3) extension:
- optimal temperature for heat-stable DNA polymerases to work
- new strand is synthesized
- 72 C
CRISPRs
Clustered Regularly Interspersed Short Palindromic Repeats
