unit 3

DNA history timeline

• meischer

• levene

• griffith

• hammerling

• oswald, McLeod, McCarthy

• Chargaff

• Hershey, Chase

• Franklin, Wilkins

• Watson, Crick

meischer

• worked in army camp and observed puss cells from discarded bandages

  • found material that very very acidic and had a large phosphorus content

  • puss cells were white blood cells, called “nuclein” (didn’t know what DNA was)

• nuclein - came from nucleus of white blood cells

levene

looked at the structure of DNA and discovered that nucleotides have phosphodiester bonds

griffith

• transforming principle

  • r strain was transformed by dead s strain material

  • genetic material got incoperated

• experiment 1

  • took s strain (smooth, protein coating) and r strain of bacteria and injected them into mice

  • s strain made mouse died

• experiment 2

  • heated s strain, destroyed protein coating. when inserted into the mouse, it lived

  • when s was combined with r, mouse died (transforming principle)

• arichibald garrod linked errors in metabolism to enzymes (proteins)

hammerling

• goal was to find where genetic material is

• cut the cap off a plant to see if it would regenerate, it did

• cut the foot off, did not regenerate

• genetic material was in the nucleus

• also found that the foot determined the color of plant (thats where the nucleus is)

avery, McLeod, McCarthy

• observed r strain and heat treated s strain (outer layer destoryed), introdced the s strain to the r strain

• wanted to see if its the protein, rna, or dna responsible for the mouse living

• when protese destroyed the protein, s cells still transformed and mouse died

• when rnase destoryed rna, s cell still transformed and mouse died

• when dnase destroyed dna and the r cells were fine and the mouse survived

  • dna causes the transformation

chargaff

discovered the 4 DNA bases and that they are complementary

hershey, chase

• experiment 1:

  • Took a virus and put a radio labelled phosopho-32 (bc dna has phosphodiester bonds)

  • Let virus infect bacteria to see if  protein or dna would be transferred to the bacteria

    • Found dna was given to the bacteria 

• experiment 2:

  • Coated the protein of the virus with sulfur (due to sulfur bonds)

  • Let virus infect the bacteria, no sulfur wnet in

  • Confirmed that the dna is transferred, not protein

franklin, wilkins

• Franklin came up with the picture, x-ray crystallography 

  • 51st draft of the pics taken, most clear (photo 51)

  • Wilkins stole the picture and gave it to other scientists, they got the nobel prize over franklin

photo 51

• Sugar phosphate backbones of DNA faced the outside of molecule, NOT the inside. 

• DNA was double helix, rotated clockwise. 

• Diameter 2 nm and one turn of helix was 3.4 nm

• 10 nucleotides every turn

• Wasn’t sure about nitrogenous bases, so didn’t publish. 

watson, crick

• used the stolen photo 51

• figured out the DNA model

gregor mendel

• “father of genetics”

• proposed that certain traits were passed from paents to offsprings

• did this with pea plants

DNA

• codes for all the instructions needed for optimal functioning of our bodies

• all cells come from a pre-existing cell

• found in nucleus for eukaryotes, cytoplasm for prokaryotes (nucleoid and plasmid)

• phosphodiester bonds in sugar-phosphate backbone

• glycosol bond between the bases and sugar-phosphate backbone

• hydrogen bonding between bases

• antiparallel strands

plasmid

• circular DNA in prokaryotes

• resistant gene for bacteria

• used for biotechnology purposes

chromosome

condensed DNA

• x pattern, two crossed over

gene

certain stretches of DNA that code for certain things

alleles

alternate/variant forms of a gene

DNA packaging

• two sister chromatides attached by a cetromere make up a chromosome

• chromosomes are wrapped around proteins (histones)

extraction buffer - strawberry lab

• consists of soap and salt

• soap: break down phospholipid bilayer

• salt: ionizes to seperate DNA from proteins

strawberry lab

• used because they are octoploid, have 8 chromsomes (lots of DNA we can view easily)

• doesn’t mattery which strawberry cells are used, since they all have the same DNA

• strawberry was first pulverized to break down the cell wall, then an extration buffer was added

why scientists extract DNA from human cells

from white blood cells

• check for recessive genes/disorders

• forensics

• blood typing

• research

• parental testing

protein synthesis in the cytoplasm

• eukaryotic organism’s genome is found within the nucleus

• proteins are synthesized by ribosomes either in the ER or cytoplasm

• DNA can’t leave the nucleus but it is needed for protein synthesis, so there are mechanisms to get the instructions out and to the ribsomes transcription and translation)

why can’t ribosomes make proteins in the nucleus

• when a protein is required by the cell, it is required in large amounts

• since there is only 1 DNA molecule in the cell, not many ribosomes could use a specifc gene at a time

  • all proteins needed by the cell would be made at the same rate

  • disadvantagous because it would reduce the cell’s flexibility in carrying out various activites

  • some cells need a lot of a particular protein, but not another

• when DNA copies itself, several ribosomes can come in and use that one

central dogma of molecular genetics

• answer to problem is messanger RNA (mRNA)

• DNA is copied as a single-stranded RNA

  • transcription

• DNA can make many RNA copies of itself and therefore send many copies out to the ribosomes in the cytoplasm

• ribosomes then translate the messengers (mRNA) into polypeptide chains, which are processed into proteins

  • translation

transcription

• copying the info encoded in DNA into mRNA

• transcribing is copying from one medium to another (DNA to RNA)

translation

• ribsomes using mRNA as a blueprint to synthesize protein compoased of amino acids

• translating is converting into a different langauge

  • langauge of mRNA is translated into language of amino acids

uracil vs thymine

ribonuclic acid (RNA)

• carrier of genetic infomation, like DNA

• 3 major cases of RNA molecules:

  • messenger RNA (mRNA)

  • transfer RNA (tRNA)

  • ribosomal RNA (rRNA)

transcription overview⭐️

• The Major Steps in Transcription are:

  • Initiation

    • RNA polymerase binds to the DNA at a specific site called the promoter, near the beginning of the gene

  • Elongation

    • Using the DNA as a template, the enzyme RNA polymerase puts together he appropriate ribonucleotides and builds the mRNA transcript 

  • Termination

    • Shortly after the RNA polymerase passes the end of a gene, it recognizes a signal to stop transcribing

• The mRNA is then released from the DNA and will eventually exit the nucleus

overview of translation ⭐️

• The major steps in translation are:

  • Initiation

    • Occurs when a ribosome recognizes a specific sequence on the mRNA and binds to that site

  • Elongation

    • The ribosome then moves along the mRNA three nucleotides at a time

    • Each set of three nucleotides codes for an amino acid

    • A tRNA delivers the appropriate amino acid and the polypeptide chain is elongated

  • Termination

    • When the ribosome reaches a special three-base nucleotide sequence that does not code for an amino acid, this provides the “stop” signal

    • The ribosome falls off the mRNA and the polypeptide chain is released

genetic code

• 20 amino acids found in proteins

• 4 bases in mRNA and DNA

• 3 nucleotides code for 1 amino acid, resulting in 64 possible combinations

  • this codes for the 20 amino acids

  • can create redundancy since many combinations can code for the same thing

codon

triplet of nucleotides (ex: ACT)

• each codes for a specific amino, 64 total but only 20 are needed (creates redundancy)

start codon

• AUG

• begins translation

• codes for the amino acid methionine

• occasionally GUG or UUG act as start codons

stop codon

• UGA

• UAA

• UAG

• end translation

genetic code is universal

• the same genetic code is used for translation in every organism except for a few (ex: paramecium)

• evidence that evolution of the code happened at such an early stage that all living organisms are descended from a common pool of primitive cells

• universality of genetic code has important ramifcations for genrtic engineering

  • since bacteria use the same code as humans, we can engineer bacteria to produce useful proteins like insulin

hypotheses for DNA replication

famous experiment done by meselson and stahl

• model 1: DNA replicates semi-conservativly (half stays the same, half is new)

• model 2: conservative, 1 double helix is the same as the parent and 1 completely new helix

• model 3: dispersive, each double helix is a hybrid

meselson and stahl’s DNA experiment

• wanted to determine how DNA replicates (used a similar method to hershey and chase)

• they labeled DNA with heavy N15 in e.coli and let it replicate so the n15 was incoperated into their DNA (DNA has nitrogenous bases, so nitrogen was used)

• transfered the N15 labeled bacteria into a medium that had N14, observed two generations of bacteria replication

  • used a centrifuge to seperate the nitrogen based on density and see how much was picked up

• found that DNA replicated semiconservatively, the bacteria had 1 DNA band with N15 and 1 with N14

DNA gyrase/toposiomeriase

enzyeme used to replieve tension and make sure there are no tangles while DNA is unwinded

• sometime it’ll cut a piece of DNA and put it back in its place

helicase

“unzipper” - opens up DNA by breaking the hydrogen bonds

replication origin

where replication starts, overall direction of replication if 5’ to 3’ (refers to carbon number of the sugar)

single stranded binding (SSB) proteins

ensure the complimentary base pairs of DNA do not rejoin during replication

building complementary strands

• prokaryotes: DNA polymerase-1 to 3 are used in replication and repair

• eukaryotes: 5 different DNA-polymerases

DNA polymerase-3

• builds complementary strands of DNA, using the template strand as a guide in prokaryotes

• requirments:

  • needs a primer to start with

  • only works 5’ to 3’

okazaki fragments

small pieces made on the lagging strand

• DNA replication is slower because the process must wait until DNA is unzipped, then primer can be place and then the new strand can be built in the 5’ to 3’ direction

• has to occur a lot since the lagging strand is being built on the parent strand with the 3’ to 5’ direction

leading strand

• the strand which uses the 3’ to 5’ template strand

• built towards the replication fork

• built faster since the complimentary strand can easily follow the 5’ to 3’ direction

lagging strand

• synthesized discontinously in short fragments in the opposite direction to the replication fork

• primers need to be continously applied as the replication fork forms along the DNA parent strand

• DNA polymerase 3 builds okazaki fragments

RNA primer

• 10-60 base pairs

• anneals (attaches) to DNA template strand, helps inititate DNA replication

  • marks initiation sequences as temporary

  • RNA primers will be removed later

• once the pimer is in place, DNA polyermase-3 can start elongation by adding free deoxyribonucleic triphosphates to the growing complementary strand

• synthesized by primase

replication bubble

forms when two replication forks are near each other

replication fork

• dna replication proceeds toward the replication fork on one strand and away from it for the other

• eukaryotes can have many replication forks, since they have so much DNA

DNA ligase

glues okazaki fragments together into one strand by the creation of a phosphodiester bond

DNA-polymerase-1

removes RNA primer and replaces them with the appropirate deoxyribonucleotides

DNA-polymerase-2

proof reads new strands and fixes mistakes (sometimes with the help of DNA-polymerase-1)

• if a mistake occurs: either enzyme functions as a exonuclease

• the repair must be made to avoid it being copied in subsequent replications

• errors missed by proofreading can be corrected by one of several repair mechanisms that operate after DNA replication

exonuclease

• DNA polymerase 1 or 2 can act as this

• enzyme backtracks to the incorrectly paired nucleotide, cuts it out, then continues adding nucleotides to the complementary strand

DNA replication

• strands are antiparallel

• at the replication origin, DNA is being opened up by helicase

  • DNA gyrase relieves tension during this process so DNA doesn’t get tangled

• since the complimentary base pairs have affinity to each other, SSB’s ensure they don’t combine and stay open during replication

• DNA polymerase-3 builds the new strand of DNA

  • primer made by RNA primase is made and attached to the parent strand of DNA

  • then the new strand of DNA is attached to the parent strand

• the leading strand is made

  • since the parent strand is 5’ to 3’, the new strand starts at 3’ and makes a complimentary 5’

  • its made quicker because it is working in the direction the enzyme needs

• the lagging strand is made

  • it needs more than one RNA primer since it’s parent strand has the direction 3’ to 5’

  • it has to wait until DNA opens up so it can make a new strand going 5’ to 3’ (okazaki fragments)

• RNA primer is removed by DNA-polymerase-1

• DNA ligase glues the okazaki fragments together

• DNA-polymerase-2 proof reads the new strands and fixes mistakes (sometimes with the help of DNA-polymerase)

deoxyribonucleoside triphosphates (dNTPs)

free bases in the nucleoplasm used by DNA polymerase-3 to build complementary strands of DNA

• A, T, C, G

energy for synthesis and joining bases

• energy released when the bond between the first and second phophate of dNTPs is broken is the enegry that drives the synthesis of the nucleotide to the elongating strand

  • dehydration synthesis

• the two extra phosphates are recycled by the cell

prokaryotic DNA storage

• don’t have membrane bound nucleus, but they have regions rich in DNA

• bacterial DNA consists almost entirely of one chromosome

• nucleoid - bacterial chromosome joined end to end to form a ring

• plasmid - small, circular sections that carry a few genes in the cytosol

  • used in biotechnology

eukaryotic DNA storage

• histones

• nucleosomes

• chromatin

histone

• every 200 nucleotides, DNA is coiled around a core group of 8 stabilizing proteins (histones)

• negatively charged DNA coiled around a positively charged protein

  • There are 4 different types of histones that make up a histone core: H2A, H2B, H3 and H4

  • A pair of each is organized into a histone core

  • A length of ~ 140 base pairs makes two turns around a histone core

• 8 histones wrapped by coiled DNA = nucleosome

nucleosome

• 8 histones wrapped by coiled DNA = nucleosome

• connected together by linker DNA

• another histone , H1, lives outside the histone core associating with the DNA when it enters and leaves the nucleosome

chromatin and supercoiling

• in the nucleus, the human genome is organized into chromosomes, which consist of a complex protein and DNA called chromatin

• one unbroken double-stranded DNA helix forms each chromosome

• the nucleotides in all the chromosomes would stretch to be 1.8 m long

• DNA is packaged even further by coiling strings of nucleosomes into cylinderical fibers (chromatin fibers)

  • also called solenoids

  • each coil of a solenoid contains 6 nucleosomes

• chromatin fold into the final chromatin structure by a higher level of coiling, supercoiling

human chromosomes

• 46, 44 somatic, 2 sec

• vary in size, somatic are organized according to this

• only a fraction is known to code for proteins

• 42,000 genes exist

variable number tandem repeats (VNTR)

• Noncoding regions are filled with VNTRs, also known as microsatellites

• These are sequences of base pairs that repeat over and over again (e.g., TAGTAGTAGTAG)

  • They vary among individuals

  • Their length varies as does their position in the genome

• Some have even been found within genes

• Huntington’s disease is associated with a repetitive sequence within a gene

telomeres

• Not all are detrimental

• Repetitive DNA is used as a defence mechanism against the shortfalls of DNA replication

• Telomeres are long sequences of repetitive noncoding DNA found at the ends of chromosomes

• they protect the cell from losing valuable genomic material during DNA replication

• Telomeric DNA protects chromosomes by binding proteins that stop the ends from being degraded and sticking to other chromosomes

repetitve DNA

also found in region of the centromeres, which play a role in cell division

pseudogenes

• within the genome

• DNA sequence thats similar to a functioning gene but doesn’t seem to express any RNA or protein

• these are homologus with known genes but are never transcribed

• thought of to be cripples copies of known functional genes

• LINEs and SINEs

long interspersed nuclear elements (LINE)

• function is unclear

• repeated DNA sequences of 5000 to 7000 base pairs in length that alternate with lengths of DNA sequences found in the genomes of higher organisms

short interspersed nuclear elements (SINE)

• function is unclear

• repeated DNA sequences of 300 base pairs in length that alternate with lengths of DNA sequences found in the genomes of higher organisms

ratio of coding to non-coding genes

• 2% to 98%

• important to know because comparitive genetics help us understand interconnections between different species

  • creates possibilites of reading our own genome in the future

  • understanding our genome can help us make informed decisions

non-coding region

• introns (23%)

• promotor

• regulatory sequences

• telomeres

• pseudogene

• variable number of tandem repeats (VNTR)

• Long interspersed nuclear elements (LINE)

• short interspersed nuclear elements (SINE)

CRISPR

• techique that allows for highly specifc and rapid modification of DNA in a genome

  • uses enzymes to cut and paste specfic segments of DNA

• issues:

  • bio-ethical. “designer” babies can be made

  • safety - technology is new so impacts on human health aren’t fully known

  • diffcult to distinguish a modified plant/insect from a regular one, could endanger biodiversity

• uses:

  • make organs for transplants from transgenic animals

  • make better crops

  • work on curing certain diseaes (hemophillia, rare liver diseases)

germ-line gene therapy

looks at altering “germ-line” cells (sperm and egg). These are cells that pass genes on to the next generation

Somatic gene therapy

looks at altering regular body cells, ones that do not get passed down to offspring. Used to treat diseases like hemophilia

epigenetics

• refers to DNA that switches specfic genes, alters expression without altering DNA sequence

  • can turn on/off/dim genes using epigenetic tags

• Ex: epigenetic tags in our muscles adjust themselves as a result of resistance exercise, allowing muscles to remember how they grow after they return to their original size

• effected by: smoking, diet, sleep, excercise

how epigenetics modify gene expression

• Modifies gene expression without altering DNA sequence

• DNA Methylation: This process adds methyl groups (a chemical cap) directly to the DNA molecule. High levels of methylation act as a "silencer," preventing transcription machinery from accessing the gene, thereby turning it off.

gene methylation in agouti mice

• In agouti mice, coat color and health are influenced by DNA methylation

• When the agouti gene is unmethylated, it is active, producing yellow, obese, and disease-prone mice.

• When the gene is highly methylated, it is silenced, resulting in brown, lean, and healthier mice.

• This effect is especially seen in Avy mice and can be influenced by environmental factors such as the mother’s diet, demonstrating how external conditions can impact gene expression.

intron

non-coding region

exon

coding region

upstream

anything before the gene is copied into mRNA

downstream

anything after the gene is copied into mRNA

promoter

• upstream

• usually has a characteristic base-pair pattern (one thats is high in A and T bases)

  • this is because A and T have 2 hydrogen bonds so its easier to break

• Since the promoter is letting the rna polymerase transcribe, if transcription is needed, it will be accessible and if its not needed it will be blocked

transcription initiation

• RNA polymerase binds to a part of the DNA molecule known as the promoter (upstream)

  • this starts the process of transcription by opening up the double helix

transcription elongation

• now that the helix is opened, RNA polymerase starts building the single stranded mRNA in the 5’ to 3’ direction

  • the strand is called the primary transcript (called this since it needs to be modified before leaving the nucleus)

  • ribonucleotides are added from the cytoplasm/nucleoplasm by making phosphodiester bonds. uses U’s instead of T’s

  • RNA polymerase uses only one of the strands of DNA as the template strand (antisense), the other is the coding strand (sense)

  • nucleoside triphosphates are added to build mRNA strand

• RNA polymerase doesn’t need a primer to start building the complementary strand

• the promoter doesn’t get transcribed

transcription termination

• mRNA strand is synthesized until the end of the gene is reached

• RNA polymerase recongizess the end of the gene white is comes across a terminator sequence (differes between prokaryotes and eukaryotes)

  • when it reaches a terminator the new strand dissociates from the DNA template strand

  • RNA polymerase is free to bind to another promoter and transcribe another gene

spliceosome

cut the introsn out and join the remaining exons together so that the coding regions are now continous to form the final mRNA

• have proteins called snRNP’s which do this

postranscriptional modifications

• must be done before primary transcript can leave the nucleus

• 5’ cap is added

  • Consists of 7-methyl guanosine, which forms a modified guanine nucleoside triphosphate

  • The cap protects the mRNA from digestion by nucleases and phosphatases as it exits the nucleus and enters the cytoplasm of the cell (protection from degradation)

  • This 5΄ cap will also help bind the mRNA transcript to the ribosome for the initiation of translation

• poly-A tail added added to 3’ end by poly-A-polymerase (polyadenylation)

• introns are cut out

  • if the introns are translated, protein won’t fold properly

  • removed by spliceosomes and then are recycled in the nucleus

• after all of this, it is mRNA which can be tranlsated by a ribosome into a protein

why aren’t transcriptional errors derimental

• splicing is very important and sometimes exons are put together in a different order, it doesn’t matter

• lots of RNA can be made, its just copies of DNA

  • DNA replication must be controlled so the mistake isn’t copied

RNA polymerase 1

transcribs rRNA

RNA polymerase 2

transcribes mRNA

RNA polymerase 3

transcribs tRNA and other short genes that are about 100 base pairs in length

transcription-translation time - pro

• don’t have a nuclear membrane

• once transcription starts, mRNA being produced can be simulatenously transcribed (coupled transcription-translation)

transcription-translation time - euk

• have nuclear membrane

• transcription must be completed so mRNA can leave the nuclear through the nuclear membrane to go to ribosomes for translation

introns - pro

• genes don’t have introns

• some archaebacteria possess them

introns - euk

genes contain introns

ribosome binding - pro

Ribosome recognizes the start of the mRNA transcript by Shine-Dalgarno sequence

ribosome binding - euk

Ribosomes recognize the 5‘ cap that was placed on the mRNA during the post-transcriptional modifications 

ribosome - pro

smaller ribosomes (70s)

ribosome - euk

• larger ribosomes (80s)

• mitochondria and chloroplasts have 70s

AUG for methionine - pro

Formyl-methionine is the first amino acid

AUG for methionine - euk

methionione is the first amino acid