SBI 4UI - Molecular Genetics

why study genetics

- cancer reasearch
- understanding genetic disorders
- genetically modified crops
- forensic science
- vaccine development

Mendel

- studied reproduction in pea plants and tracked traits over generations
- determined "unit characteristics" were the method of passing on traits for inheritance

Miescher

- studied nuclei of pus cells from discarded surgical bandages
- detected a phosphorus-containing substance that he named nucleon
- first one to isolate DNA (didn't know what it was but called it nucleon because it was from the nucleus)

Griffith

- studied S-strain (virulent) and R-strain (nonvirulent)
- heat-treated S-strain didn't kill mice
- heat treated S-strain + R-strain killed mice
- came up with transformation

transformation

a change in genotype/phenotype by the direct uptake of genetic material by a cell

Hershey & Chase

- used a virus to infect a bacterial cell
- used P32 to label DNA of the virus
- used S35 to label proteins of the virus
- radioactivity was only detected within bacteria cells with viruses containing DNA labelling
- found that DNA functions as the genetic material

Wilkins & Franklin

- used x-ray crystallography to find the pattern of DNA

Wilkins & Franklin didn't get along...

Wilkins first used this that suggested DNA had a helical structure but Franklin disagreed
- his DNA samples were poorly prepared so his graphs were difficult to interpret
Franklin tried again
- she prepared a purer DNA sample and produced clearer graphs that showed the pattern of a X
- after analysis, she suggested that the sugar-phosphate backbones faced the outside - not the inside- and that DNA was a double helix that rotated in a clockwise direction
- she determined that DNA had a diameter of 2nm and one turn of the helix was 3.4nm in length

Watson & Crick

- incorporated everything they new about DNA to make a model that shows the actual structure of DNA

Chargaff

the individual responsible for discovering the base pairing rules for DNA

heredity

process that allows genetic information to be passed from one generation to the next

DNA

double stranded helical molecule that contains hereditary information

chromatin

strands of DNA found in the nucleus, can be condensed using histone proteins

eukaryotic chromosomes

condensed forms of chromatin that exits during cell division, found in eukaryotic cells and have a X shape

bacterial chromosomes

- always exists
- looped om a circle
- DNA is always in chromosomes because there is no nucleus to protect the chromatin

gene

a small section of DNA that codes for/directs a specific cellular function

allele

a version of a gene

histones

a protein using to coil and protect chromatin in the nucleus

plasmids

- small circular sections of DNA found in bacteria, usually important in resistance

genome

full set of genetic information for an organism

DNA - structure

- polymer arranged as a double-stranded helix
- strands run antiparallel
- monomer subunits called nucleotides

nucleotides

three components:
- pentose sugar
- phosphate group
- nitrogenous base (A,T,C,G)

purines

- adenine & guanine
- two rings

pyrimidines

- thymine and cytosine
- one ring

adenine & thymine make

- two hydorgen bonds

cytosine & guanine make

- three hydorgen bonds

phosphodiester bond

- intramolecular bond that holds sugar-phosphate backbone together
- covalent bond
- strong bond

hydrogen bond

- intermolecular bond that holds nucleotide bases together
- weak bond, but there are millions which allow for DNA to be molecularly strong
- broken during DNA replication

glucosyl bond

- intramolecular bond that holds base to the sugar
- holds nucleotides to the backbone

guanosine triphosphate (GTP)

- like ATP but with guanine not adenine

cyclic adenosine monophosphate (cAMP)

- formed after the removal of both high energy Pi's
- after chemical signal binds to cell surface, it triggers the conversion of ATP to effects inside the cell
- like ATP but it is cyclical

three theories were proposed based on DNA replication

- conservative
- semi-conservative
- dispersive

conservative

- entire double helix replicated at one time

semi-conservative

- two strands that make up DNA pull apart at the H-bonds and two new complementary strands from against each one

dispersive

- DNA breaks into nucleotide pieces and new and old re-form

Melelson and Stahl

- grew DNA in a heavy nitrogen 15
- then transferred them to a lighter nitrogen 14
- saw that after awhile the DNA was a medium weight then over more time became light
- thus proving semi-conservative theory was right

replication origin

- where replication begins

topoisomerase

- relieve tension by cleaving the DNA to allow for the unwinding of the double helix
- keeps the strand from spinning back together

DNA helicase

- breaks hydorgen bonds between base pairs to unwind the DNA

single-stranding binding proteins (SSBP)

- keep seperated strands of DNA apart y blocking hydorgen bonding

RNA primase

- needed to start the chain
- adds about 10 RNA nucleotides to provide a place where DNA polymerase III can start

DNA polymerase I

- replaces RNA primer nucleotides with DNA nucleotides

DNA polymerase III

- an enzyme that adds nucleotides to the chain

two different strands of DNA

- leading strand (built continuously)
- lagging strand (built in many different sections, requires more enzymes to glue sections together)

DNA replication always works from

3' to 5'

DNA replication

- replication begins at an origin and causes bubbles to open - makes a replication fork
- topoisomerase relieves tensional forces
- DNA helicase unwinds the double helix
- DNA strands pull apart, as the 3' end opens the replication of the strand begins - called the leading strand
- SSBP keeps the helices apart

DNA replication - leading strand

- nucleotides can only be added to the 3' end (made from the 3' to 5')
- RNA primase adds primer to beginning of the strand first
- DNA polymerase III adds nucleotides continuously on the leading strand

DNA replication - lagging strand

- once some of the molecule is pulled apart the lagging starts to form
- made from 5' to 3' and is non-continuous
- RNA primase adds primers - nucleotides are added in short Okazaki fragments by DNA polymerase III
- DNA polymerase I replaces RNA primers from fragments
- DNA ligase glues the fragments together into one strand by creation of a phosphodiester bond
- as complementary sequences are built, DNA polymerase III and I proofread the new strand
- exonuclease fixes mistakes that DNA polymerase I and III finds

telomeres

- non-coding repeating sequences of DNA
- protect the coding regions of DNA
- prevent chromosome ends from fusing
- may help determine life span of organisms and aging
- over time as you age you loose a bit of telomeres
- created because DNA replication does not replicate all of it - some is left behind

codon

- three base pair that codes for all proteins in our bodies

replication & transcription occurs in the...

nucleus

translation occurs in the...

cytoplasm (attached to ribosomes)

stages of transciption

- initiation
- elongation
- termination

DNA transcription

- used to convert DNA to messenger RNA (mRNA)

mRNA processing in prokaryotes

- no mRNA processing

mRNA processing in eukaryotes

mRNA processing which includes:
- a methyl cap
- poly-A tail
- introns removal

DNA transcription - initiation

- RNA polymerase attaches to DNA at a specific point called the promoter region (TATA box)
- RNA polymerase unwinds and unzips DNA exposing the template strand but doesn't proof read it

DNA transcription - elongation

- mRNA is synthesized using the 3' to 5' strand of DNA as a template
- RNA polymerase moves along DNA synthesizing mRNA in the direction of 5' to 3'
- thymine is changed to uracil
- RNA polymerase reaches termination sequence at the end of the gene and stops transcription
- RNA is never all the way unzipped, only enzymatic activity in certain sections

DNA transcription - termination

- RNA synthesis stops when RNA polymerase reaches a termination sequence
- mRNA and RNA polymerase are released from the DNA strand
- DNA zips back together and can be reused again

mRNA processing in eukaryotes

- cells have nucleases in cytoplasm must therefore be protected to allow for protein synthesis to occur

two mechanisms needed to protect the mRNA

- 5' cap protects it from degradation
- poly-A tail is added to the 3' end by poly-A polymerase

spliceosomes & snRNP

- helps remove introns
- snRNP causes a loop to from and remove the intron & join the exons
- when the loop is created this activates the spliceosome
- spliceosome are enzyme/protein complexes that remove introns and join exons together
- intron loop is released and degraded
- snRNP's are released and reused

messenger RNA (mRNA)

- varies in length, depending on the gene that has been copied
- acts as the intermediate between DNA and ribosomes
- translated into proteins by ribosomes
- is the RNA version of the gene encoded by DNA

transfer RNA (tRNA)

- functions as the delivery system of amino acids to ribosomes as they synthesize proteins
- is very short, only 70-90 bases long

ribosomal RNA (rRNA)

- bonds with proteins to from the ribosomes
- varies in length

central dogma

- fundamental idea of molecular genetics which say that genetic information flows from DNA to RNA to proteins

location of transcription prokaryotes vs. eukaryotes

prokaryotes:
- throughout the cell
eukaryotes:
- in the nucleus

enzymes of transcription prokaryotes vs. eukaryotes

prokaryotes:
- single type of RNA polymerase transcribes for all types of genes
eukaryotes:
- different RNA polymerase are used to transcribe genes that encode protein (RNA polymerase II) and gene that do not encode protein (RNA polymerase I,III)

elongation of transcription prokaryotes vs. eukaryotes

prokaryotes:
- bases are added quickly
- 15 to 20 nucleotides per second
eukaryotes:
- bases are added slowly
- 5 to 8 nucleotides per second

promoters of transcription prokaryotes vs. eukaryotes

prokaryotes:
- less complex than those in eukaryotes
eukaryotes:
- immediately upstream of protein-coding genes, and they are more complex than those in prokaryotes

termination of transcription prokaryotes vs. eukaryotes

prokaryotes:
- protein binds to the mRNA and cleaves it, or the mRNA binds with itself
eukaryotes:
- nuclear proteins bind to the polyureic site and terminate transcription

introns and exons of transcription prokaryotes vs. eukaryotes

prokaryotes:
- there are no introns
eukaryotes:
- there are both introns and exons

product of transcription prokaryotes vs. eukaryotes

prokaryotes:
- results in mRNA ready to translated into proteins by ribosomes
eukaryotes:
- results in pre-mRNA, which must be modified to protect the final mRNA degration in the cytosol and to remove introns

mRNA translation

- converting mRNA to proteins
- faciliated by transfer RNA 9tRNA)
- occurs in the ribosome within the cytosol

three stages of translation

- initiation
- elongation
- termination

tRNA

- produced in the nucleus using the same process as mRNA
- assumes a 3D shape that looks like a clover
at least 20 different tRNA's (one for each amino acid)
- picks up specific amino acids from the cytosol

anticodon

- recognizes the codon of the mRNA
recognition is faciliated by the complementary base pairs (allows for the amino acid to be bound to the proper spot)

aminoacyl-tRNA

- tRNA molecule with its corresponding amino acid attached to its acceptor site at the 3' end
- amino attached to the tRNA

aminoacyl-synthetase

- enzyme hat adds the correct amino acid to each tRNA
- at least 20 of them (one for every amino acid)
has two active sites:
- one recognizes the anticodon on tRNA molecule
- the other recognizes amino acid corresponding to that anticodon

ribosomes

- made up of two subunits
- brings together the mRNA strand and the aminoacyl-tRNA and the enzymes involved in building polypeptides
- moves mRNA in the 5' to 3' direction

A site

- acceptor site
- holds amino acid tRNA that awaits for its amino acid to be added to the growing chain

P site

- peptide site
- where peptide bonds are fromed and holds the growing amino acid chain

E site

- exit site
- releases tRNA back into cytoplasm

translation - initiation

- small subunit of ribosome attaches to the 5' cap of mRNA and is skipped over until AUG sequence is found and initiation occurs
- AUG codes for methionine so every protein starts with methionine

stop codon

- a codon that signals the end of a polypeptide chain and causes the ribosome to terminate translation

initiation codon

- the codon that signals the start of a polypeptide chain and causes the ribosomes to start translation
- AUG

translation - elongation

- the first codon (AUG) is recognized by an anti-codon (UAC) on tRNA
- the tRNA enters the ribosome at the A site, bring an amino acid along with, then it moves to the P site
- peptide bonds are formed between the junction of the P and A site
- enegry is provided by the hydrolysis of GTP
- tRNAs then move to the E-site, where they exit
- process repeats itself until the ribosome reaches a stop codon (UGA, UAG, UAA)

translation - termination

- when a stop codon on the mRNA enters the A-site, there is no corresponding tRNA for that codon
- a release factor bonds to the stop codon in the A site and dismantles the ribosome/mRNA complex

primary protein structure

- determined by amino acid sequence
- one long chain

secondary protein structure

- results from hydrogen bonding between amino acids
- α helix (coiled), β-pleated sheet (folded) shapes

tertiary protein structure

- helixes and B-sheets fold upon themselves
- supercoiling 3D structures
- disulfide bridges from between amino acids

quaternary protein structure

- two or more separate polypeptide chains interacting

post-translational modifications (3D)

- polypeptide folds into its 3D shape creating secondary and tertiary structure
- polypeptides may be joined together creating quaternary structure
- chaperone protein may help with folding

post-translational modifications (adding things)

- glycosylation occurs when sugars are added
- other enzymes may remove amino acids or cleave polypeptide chain into 2+ pieces
- phosphorylation occurs when phosphate groups are added

step 1 of protein synthesis

DNA unwinds

step 2 of protein synthesis

mRNA copy is made from one of the DNA strands step 1 of protein synthesis

step 3 of protein synthesis

mRNA copy moves out of nucleus into cytoplasm

step 4 of protein synthesis

tRNA molecules are activated as their complementary amino acids are attached to them

step 5 of protein synthesis

mRNA copy attaches to the small subunit of the ribosomes in cytoplasm and six of the bases of the mRNA are exposed in the ribosome

step 6 of protein synthesis

a tRNA bonds complementarity with the mRNA via its anticodon