bio ch 16 `17

purpose, general protocol, and results of griffith's experiment

purpose: griffith wanted to figure out what bacteria causes diseases
general protocol: injected two types of bacteria into a mouse (rough and smooth) and observed results
results:
- found that it was fine with a rough and heat-killed smooth, but not with heat-killed smooth mixed with the rough
- concluded that there was there was a transforming factor that was transferred from dead smooth strain to live rough strain and making it virulent

purpose, general protocol, and results of avery, mccarty, and macleod's experiment

purpose: tried to figure out what the transforming factor in griffith's experiment was
general protocol: broke down bacteria and then isolated RNA, proteins, and DNA to break them down and see if they could transform the bacteria again
results: the only time bacteria couldn't transform when they broke down DNA, proving DNA was transforming factor (people were still skeptical though)

purpose, general protocol, and results of hershey-chase's experiment

purpose: to definitively conclude that it was actually DNA that was the transforming factor or proteins in DNA
general protocol: used bacteriophages in sulfur (in proteins, not in DNA - dyed red) and phosphorous (in DNA, not in proteins - dyed green) and see which did the transforming
results: the one with the green dye (phosphorous) was the only one transforming, definitively proving that DNA was transforming factor

purpose, general protocol, and results of meselson and stahl's experiment

purpose: try to determine the process that DNA was replicated
general protocol: they grew E. Coli in a medium contining heavy nitrogen (N-15) and then added the E. Coli to a medium containing regular nitrogen (N-14) and observed it over several generations
results: proved that DNA was replicated semi-conservatively

contributions of chargaff

-base pair rule: 1:1 ration of nitrogenous bases
-guanine is equal to cytosine, adenine is equal to thymine
- known as Chargaff's rule

contributions of watson + crick

discovered the double helix structure and dna's backbone

contributions of wilkins + franklin

took an x-ray crystallography picture of DNA that helped show the double-helix shape of DNA (watson got idea from meeting with franklin)

evidence of common ancestry in dna

genetic code is universal (reason retroviruses work)

structure of dna

- double stranded
- strands run anti-parallel in helix shape
- backbone is made of 5-carbon deoxyribose and phosporous
- covalent phosphodiester bonds between sugars and phosphates
- hydrogen bonds between nitrogenous bases

nitrogenous bases

adenine, guanine, cytosine, thymine, uracail

pyrimidines

cytosine, thymine, uracil (single ring)

purines

adenine and guanine (2 rings)

purine-pyrimidine bonding

2 hydrogen bonds between A-T and A-U
3 hydrogen bonds between G-C

nucleotide composition

5 carbon sugar, phosphate group, nitrogenous base

location of replication

in eukaryotes: the nucleus
in prokaryotes: the cytoplasm

three types of replication

- conservative: one helix made of completely old dna and one made of completely new dna
- semi-conservative: two helixes both with one strand of old dna and one of new dna
- dispersive/disruptive: old dna breaks into fragments and the strands contain both old and new dna

differences in replication in prokaryotes compared to eukaryotes

prokaryotes: only one origin of replication, replication happens in two opposing direction at same time, and is in cytoplasm
eukaryotes: multiple points of origin, replication happens in one direction, in nucleus

why do eukaryotes have multiple points of origin?

because its chromosomes are very long so you need multiple in order for replication to happen quickly

replication fork

y-shaped region where the parental strands of DNA are being unwound

steps of dna replication

1. Helicase unwinds the DNA
2. Topoisomerase relieves any twists formed in the DNA as a result of unwinding
3. Single stranded binding protein prevents reattachment of each parental strand to each other
4. Primase initiates DNA replication at origins of replication, using RNA primers
5. DNA polymerase attached to the RNA primers and begins the process of elongation
6. DNA nucleotides replace RNA primers
7. The leading strand is continuously synthesized
8. The lagging strand is synthesized as short Okazaki fragments
9. DNA ligase joins Okazaki fragments

enzymes and molecules required for dna replication

- helicase
- ss binding protein
- dna polymerase
- topoisomerase
- primer
- primase
- dna ligase

helicase

enzymes that untwist the double helix at the replication forks, separating the two parental strands and making them available as template strands

single-strand binding proteins

bind to unpaired DNA strands, keeping them from re-pairing

topoisomerase

helps relieve this strain by breaking, swiveling ,and rejoining DNA strands

primer

initial nucleotide chain that is produced during DNA synthesis made of RNA; generally 5-10 nucleotides long

dna primase

enzyme that intiates dna synthesis by creating a primer

dna polymerase

- catalyzes the synthesis of nucleotide by adding nucleotides to a preexisting chain
- replaces rna nucleotides to be the primers with dna versions

dna ligase

joins okazaki fragments together

leading strand

strand that is read from 3' to 5' that elongates continuously because new nucleotides are added on the 3' end of primers (only one primer is needed)

lagging strand

strand that is read 5' to 3', making the polyermase work away from replication fork, so it is synthesized in segments (primer is needed for each segment since polymerase has to detach and reattach at each segment)

okazaki fragments

segments of lagging strand named after japanese duo that discovered them

three types of dna-repair mechanisms

- base-excision repair
- nucleotide-excision repair
- mismatch pair

DNA ligase in DNA repair

joins the broken dna fragments back together

base-excision repair

when something is fundamentally wrong with the base itself (i.e. uracil in dna or bases are chemically modified)

glycosylases

special enzymes that come and pluck out the base and put the correct base and seal it back in

nucleotide-excision repair

process of removing and then correctly replacing a damaged segment of DNA using the undamaged strand as a guide

thymine-to-thymine dimer

when two thymies next to each other bond to each other and create a bucking of DNA molecule because of double bond (example of need for nucleotide-excision repair)

UVR enzymes

get rid of thymine dimers; called uvr because thymine dimers are usually due to excessive uv radiation

mismatch repair

base gets mismatched with the wrong pair and dna polymerase does not pick it up as it usually does

exonucleases

enzymes that cleave out a chunk of dna and fix the mismatch

role of dna polymerase in mismatch repair

usually dna polymerase is a great proofreader and will go back and change any mistakes but does not work in this case

mismatch repair system

- occurs at the end of replication in case the dna polymerase hasn't picked up the mismatch
- knows which is the original strand and not based on if there was an extra methyl added to a base

central dogma of biology

flow of genetic information is from dna to rna to proteins

exception to central dogma

retroviruses (go rna to dna to rna to proteins)

general sequence of integration of retroviral DNA into host cell and expression of viral proteins

Retroviral RNA🡪 viral DNA🡪 viral DNA replication🡪 viral DNA integration into host
chromosome🡪 use host cell machinery to transcribe and translate Retroviral DNA to
express retroviral genes and synthesize retroviral proteins

difference between transcription and translation

transcription : step of taking dna to rna
translation: step of taking rna to proteins

location of transcription and translation in eukaryotes and prokaryotes

prokaryotes: both in cytoplasm
eukaryotes: transcription in nucleus and translation in cytoplasm

steps of transcription

initiation, elongation, termination

enzymes involved in translation

ribosomes

initiation

rna polymerase makes a pre-mRNA transcript based on a sequence of dna

transcription bubble

section of dna double strand that gets opened up from rna polymerase for initiation of transcription

template strand

- strand that rna polymerase reads in 3' to 5' direction
- strand that is used to build pre-mRNA transcript off of
- mRNA is transcribed 5' to 3'

reason non-template strand is called a coding strand

because its code will match up with the pre-mRNA letters except for U replacing T

different names for template strand

antisense, noncoding, minus strand

different names of non-template strand

sense, coding, plus strand

transcription factors

help RNA polymerase attach to promoter sequence to help turn on or off transcription; regulation in eukaryotes

promoter

- binding site for rna polymerase
- lies upstream of the gene
- controls transcription and where it starts
- contains the TATA box and is around 25 nucleotides

TATA box

a part of the promoter that is a sequence that tells the RNA polymerase where to bind and start the process of transcription and the code is TATAAAA on the CODING strand

elongation

- nucleotides are added to 3' end of growing RNA molecule
- RNA polymerase elongates 5' to 3' and the DNA template strand is read 3' to 5'

termination

rna transcription gets cleaved away from dna and left with completed pre-mRNA transcript

polyadenylation signal

sequence of letters that act as a termination signal for rna polymerase and 10-35 nucleotides, termination happen (code: AAUAAA)

important process that happen in mRNA processing

splicing and modifications

mRNA processing

- occurs in nucleus of eukaryotes
- happens because mRNA must be processed before it can enter the cytoplasm

modification that happen during mRNA processing

- 5' cap: made of 7-methyl-guanosine which is a cousin of adenosine
- poly-A tail: modification on 3' end where a bunch of adenines get added to help prevent mRNA from getting degraded too quickly

reasons for mRNA processing

1. helps stabilize mRNA (protect it from degradation)
2. helps in actual export of mRNA through pores
3. increase translational efficiency
4. adds to enormous genetic diversity

splicing

- process of where pre-mRNA transcript gets made into mature RNA
- introns get removed
- exons remain

role of spliceosomes

group of snRNP's that are used to splice out introns by recognizing splice sequences to cut

alternative splicing

produces variety of mRNA transcripts based on which exons are kept and which leads to variety of polypeptide chains

ribozymes

- RNA molecules that act as enzymes and splice their own introns (self-splicing)
- discovery proved that not all enzymes are proteins

steps of translation

initiation, elongation, termination

structure of ribosome

- small subunit
- large subunit
- EPA sites
- made of rRNA and proteins

how ribosomes interact with mRNA

- its structure reflects its function of bringing it together with tRNA
- it acts as a binding site for mRNA

initiation of translation

translation starts with a start codon (AUG) which activates the ribosome and the first tRNA molecule enters at the P-site

elongation of translation

another tRNA molecule enters the ribosome at A-site when the ribosome continues reading the mRNA transcript and the first tRNA molecule will move to the E-site, leaving its amino acid behind the P-site

termination of translation

- begins with a stop/nonsense codon and causes a release factor, which enters at the A-site and causes the small and large subunits to disassemble
- polypeptide chain is released and sent to golgi for further processing and folding and mRNA goes back to nucleus so it can be broken down and reused

relationship between codons, anticodons, and amino acids

- mRNA matches with an anticodon, which is on a tRNA molecules, which contains the amino acid that the codon actually codes for
- anticodon is read/run in opposite direction of codon (5' to 3' \= codons, 3' to 5' \= anticodons)

why is a codon made up of 3 bases

because if a codon were made of 2 bases, that would only code for 16 amino acids and it allows for wiggle room

dna triplet vs mRNA codon vs tRNA anti-codon

dna triplet: same as mRNA codon but DNA letters
codon: set of 3 RNA bases that determine the polypeptide chain made
anti-codons: set of 3 RNA bases complementary to the codons and are attached to the amino acids the codons code for; prevents delivery of wrong amino acids to ribosome

what to look at when determining the sequence of amino acids

look at codon not anti-codons

how many codons, start codons, and stop codons are there?

64 codons
1 start codon (AUG - methionine)
3 stop codons (UAA, UAG, UGA - no amino acid)

structure of tRNA molecule

- four base-paired regions
- three loops
- amino acid attachment site
- anticodons on the bottom loop

type of bond of bases in tRNA

hydrogen bonds

type of bond between amino acids on tRNA mols

covalent bonds

wobble hypothesis

- exception to watson-crick base pairing on position 3 of codon and position 1 of anticodon
- provides wiggle room for any unusual pairing/mutation on the third position and ensures the amino acid won't change

mutations can be

+, -, or neutral depending on environment

gene mutation

look at a singular gene mutation; can lead to a change in the type or amount of protein produced

types of gene mutations

point and multiple base pair

point mutation

pertains to one base

substitution mutation

type of point mutation where you substitute of one base of another

types of substitution mutations

missense: substitution changes amino acid
nonsense: substitution leads to stop codon
silent: substitution does not change amino acid

frameshift mutation

type of point mutation where you add or delete one base pair; generally more dangerous than base substitutions

results of frameshift mutations

- all other bases shift down from where the base was deleted or inserted and can mess up every other amino acid that follows
- can result in missense and nonsense mutations
- cause of many genetic disorders (ex: Tay-sachs)

multiple base pair mutations

subsitution, insertion, or deletion of more than one base pair

least dangerous version of frameshift mutations

when you add multiples of three because you're just adding an extra amino acid instead of potentially changing every amino acid for the rest of the polypeptide chain

chromosomal mutations

can occur to multiple different genes and usually happen during meiosis; mutations are passed down in gametes but not in somatic

types of chromosomal mutations

- gene duplication
- translocation
- inversion
- deletion

gene duplication

results from unequal crossing over, multiple copies

translocation

piece of one chromosome dislodges and is attached to nonhomologous partner

inversion

order of genes on that section of chromosome get switched up

some significant problems of base substitutions

sickle cell anemia