Biology 30 AP - DNA & Protein Synthesis

History of DNA

1952 - Rosalind Franklin → X-ray diffraction shows helical structure of DNA

1953 - Watson & Crick → publish paper on complete double helix structure of DNA

• used Franklin’s photograph (Photograph 51) of DNA and claimed it as their own

Where is DNA found in plants?

nucleus

chloroplast

mitochondrion

What is unique about the way mitochondrial DNA is passed down?

only inherited from the mother (thousands in the egg compared to the sperm)

DNA structure

double helix (twisted ladder)

each unit made of sugar (deoxyribose) and phosphate backbone with nitrogen base (“rungs”) held together with covalent bond (a.k.a. nucleotide)

nitrogen bases from one backbone connects to complementary nitrogen base on another backbone via hydrogen bonds

strands are put together anti-parallel → one strand is “upside-down” compared to other

Four nitrogen bases

adenine (A)

guanine (G)

thymine (T)

cytosine (C)

Two types of bases

purines (double ring structure) - A & G

pyrimidines (single ring structure) - T & C

How do the bases pair with one another?

adenine always forms bonds with thymine (A—T): 2 HB

guanine with cytosine (G—C): 3 HB

purines pair with pyrimidines

Strongest intramolecular bonds

within a nucleotide

covalent bonds

Strongest intermolecular bonds

between bases

hydrogen bonds

Chargaff’s Rule

amount of A=T

amount of C=G

DNA replication

DNA must be replicated for growth and reproduction

cells must have nucleotides available → from food

semi-conservative → new double helices each contain one original (parent) strand and one new (daughter) strand

number of enzymes required

Unwinding enzymes

unzip double helix

Primase

initiate replication site by adding primers

Polymerase

add new nucleotides

remove primer

checks for mistakes

Ligase

glues fragments together when synthesis complete

Mutations

unrepaired mistakes

rate increases with exposure to mutagens (toxic chemicals, radiation)

germ cells (eggs/sperm) mutation → passed on to every cell of offspring

mutations may cause cancer and produce more viable offspring (increased variation)

Steps of DNA replication

1. Helicase unwinds/unzips DNA

2. Binding proteins keeps DNA unzipped

3. Primase adds RNA primers

4. Polymerase III adds bases in 5’ to 3’ direction starting at primer

• leading strand → continuously towards replication fork

• lagging strand → Okazaki fragments away from replication fork

5. Polymerase I removes RNA primer and replaces with DNA nucleotides

6. Ligase glues new DNA

7. Polymerase III fixes mistakes

Gene

functional sub-unit of DNA that directs production of one or more polypeptides (protein molecules)

not spaced regularly among chromosomes

information converted into a specific characteristic/trait through polypeptide production

order of the base pairs in a DNA molecule makes up the genetic code of an organism

Genome

sum of all DNA that is carried in each cell of an organism

includes genes and regions of non-coding DNA

What is there no set relationship between?

number of genes in an organism and total size of its genome

Polypeptides

made up of amino acids → 20 amino acids → proteins made up of polypeptides

one gene → one protein → up to 50 000 amino acids = gene expression = protein synthesis

How many bases are needed to code for all 20 amino acids?

if 1 - only 4 amino acids could be coded (A,C,T,G)

if 2 - only 16 amino acids could be coded (4×4)

if 3 - up to 64 amino acids can be coded (4×4×4)

Codon

group of 3 bases

Central dogma

eukaryotic cells

DNA → mRNA → protein

transcription → translation

DNA vs. RNA

copy of gene required to synthesize specific protein is made in nucleus → mRNA (messenger RNA)

one strand of the DNA is copied into RNA → similar to DNA synthesis except:

• much shorter strand

• single-stranded

• no thymine → uracil pairs with adenine (AUGC)
  sugar backbone made from ribose

• RNA polymerase is used to make mRNA

Transcription

promoter and terminator sequences (before and after gene) tell RNA polymerase where to start and stop

once mRNA is made, it is processed

introns (non-coding regions) removed (“incised”)

leaves only exons (“expressed”) as template

mRNA moves to cytoplasm → protein synthesis occurs at ribosomes

Coding sense strand

5’ to 3’

carries the translatable genetic code and has the same sequences as the mRNA

Non-coding/template anti-sense strand

3’ to 5’

template for producing mRNA which directs the synthesis of a protein

complementary to the RNA and acts as a guide

How does RNA polymerase work?

1. Binds to promoter on template strand (promoter sequence comes before the gene and tells the RNA polymerase where to start)

2. Section of double helix opens (initiation)

3. RNA Polymerase II moves along DNA and completes a strand of mRNA complementary to DNA (elongation)

4. Copies the DNA in 5’ to 3’ direction

5. Adds one nucleotide at a time (uracil - U - instead of thymine - T)

6. Reaches a terminator sequence that signals transcription to stop (termination)

7. mRNA released and travels to cytoplasm

8. DNA recoils

Summary of genetic code

redundant → multiple codons can code for the same amino acid

continuous → no spaces between codon = mistakes can compromise entire protein

(nearly) universal → almost all organisms build proteins with same codons & aa combos

Translation

interpretation of mRNA to make protein takes place on ribosomes (protein/RNA complex) in cytoplasm

mRNA attaches to ribosome → tRNA (transfer RNA) brings amino acids to complex → polypeptide chain assembled

tRNA (transfer RNA)

two specific ends

one that reads the mRNA triplet (anticodon)

one that attaches to a specific amino acid

Anticodon

complementary to mRNA codon

Ribosomes

small organelles made mainly of rRNA (and some proteins)

made of a large and small subunit

match tRNA with the corresponding mRNA

brings in enzymes to form bonds between amino acids

free ribosomes → synthesize proteins for use in the cell

RER → synthesize proteins for excretion

Initiator/start codon

all proteins start with it

AUG → always methionine

Terminator codon

all proteins end with it

UAA, AUG, or UGA (stop codons)

Steps of translation

1. mRNA binds to ribosome so that two codons are exposed

2. tRNA with methionine is first to bind

3. second tRNA (to match second codon) brings in second amino acid

4. enzyme catalyzes peptide bond formation

5. ribosome moves one codon along chain and process repeats

6. continues until stop codon reached

7. at stop codon, chain is released and ribosome complex disassembles

Mutation

permanent change in genetic material of an organism

can occur in somatic or germ cells

Somatic cells

mutations in non-sex cells that are not passed on to next generation → can lead to cancer

Germ cells

mutations in reproductive/sex cells → passed on from one generation to the next

Point mutation

affects one or few nucleotides

Substitution

one nucleotide for another

3 types: missense, silent, and nonsense

Frameshift mutations

insertions (additions): an extra base is slipped in

deletions: a base is missing

not multiples of three

Missense mutation

results in an altered protein (e.g. sickle cell anemia)

Silent mutation

has no effect on a cell’s metabolism

Nonsense mutation

renders gene unable to code for a functional polypeptide

Causes of mutations

spontaneous → DNA polymerase incorrectly pairing nucleotides

induced by a mutagen

3 types of mutagens

most are carcinogenic (cancer-causing)

physical: forcibly break a nucleotide sequence, causing random changes (e.g. X-rays, UV radiation)

chemical: enters cell nucleus and causes permanent change in genetic material by reacting chemically with DNA (nitrites)
infectious: bacterial or viral pathogen

Is mutation always bad?

frameshift mutations can result in whole new protein → rarely good for an organism

can sometimes generate new, beneficial trait (genetic variability)

Genetic engineering

science of manipulating genes that carry hereditary information

Recombinant DNA Technique

1. Use a restriction enzyme to cut a piece of DNA from selected organism to isolate the desired gene

2. Use the same restriction enzyme to cut vector DNA

3. Insert DNA (gene) into vector genome

4. Glue pieces together with ligase. Allow recombination

5. Have hope the cells take up the DNA

6. Vector will replicate, transcribe, and translate inserted gene along with rest of organism’s genome

7. Use a selection technique to determine if DNA has recombined the way you want it to in vector

Selection technique

include a gene for antibiotic resistance beside the gene you really want

try to grow the vector/bacteria in that medium → only those bacteria that have incorporated the DNA you want will be able to grow

clone the bacteria

Vectors

provide a means to get genes where you want them → viruses, bacteria, plasmids (circular and self-replicating)

Restriction enzymes

recognize specific sequences and cut DNA into pieces with uneven ends → “sticky ends”

Ligase

used to rejoin sticky ends of DNA cut with restriction enzymes

Targeted gene expression

insert human genes into other organism’s genome that cause some human traits to be expressed (e.g. HGH, insulin)

Gene therapy

providing “fixed” genes to people with faulty genes (must use a vector)

Biological warfare

insert harmful genes into harmless bacteria → transfer to food/water → mass infections with resistant bacteria

3 goals of the Human Genome Project (1990-2003)

determine the location of all of the genes in the human genome

sequence the bases in the human genome

determine the function of the genes

DNA fingerprinting

copied (by polymerase chain reaction - PCR) and cut DNA is separated using gel electrophoresis

Gel electrophoresis

electric current run through gel

negatively charged DNA attracted to positive cathode

larger strands move slowest

branding pattern appears