IB Bio HL Unit 7

DNA replication

the process of copying the genome within a cell. creates 2 identical copies with 2 complementary strands

Meselson and Stahl experiment

experiment to determine how DNA was replicated, concluded it is semi-conservative

semi-conservative

after DNA replication, the new helix as one strand of old DNA and one strand of new DNA

how is old DNA used in replication?

double stranded DNA unzips so each strand acts as a template for new DNA

how are genetically identical molecules produced?

the complimentary base pairing ensures that each DNA molecule is identical

helicase

an enzyme that binds to the origin of replication, unzips the double helix by breaking hydrogen bonds between bases

origin of replication

region of DNA where replication begins

replication fork

the region where OG DNA double helix splits into 2 strands

single strand binding proteins

bind to single stranded DNA to keep strands separate by preventing hydrogen bonds from reforming

gyrase

moves in front of helicase to relieve tension caused by supercoils created by helicase unzipping DNA

DNA polymerase III (DNA pol III)

the enzyme that reads the template and builds complimentary strands, builds in the 5’→3’ direction. can only add new nucleotides to an existing strand at the 3’ end.

why does DNA pol III only work in one direction?

[—] is an enzyme with an active site that is specifically shaped, so it only builds new strands in the 5’→3’ direction

primase

an enzyme that creates an existing strand for DNA pol III to add to the 3’ end

primer

a sequence of RNA nucleotides added to existing strands of DNA pol III which begins creating a complementary strand

DNA proofreading

DNA pol III proofreads newly formed DNA strands as it is being built, removing and replacing mistakes. some mistakes are missed!!! (mutations)

DNA polymerase I (DNA pol I)

this enzyme removes RNA nucleotides (primers) and replaces them with the correct DNA nucleotides

leading strand

this strand is synthesized continuously by DNA pol III and follows the same direction of the helicase (5’→3’)

lagging strand

this strand is synthesized discontinuously away from the replication fork

how does replication work in the leading strand?

only 1 primer is required to start replication, and once it’s created DNA pol III follows the helicase until the molecule has been unzipped.

how does replication work in the lagging strand?

this strand is replicated with multiple primers and Okazaki fragments by DNA pol III moving away from replication fork

Okazaki fragments

the fragments sectioned off on the lagging strand. each needs its own primer!

enzyme activity in lagging strand

this strand has more primase and DNA pol I activity: 1 primer for each Okazaki fragment, therefore more primers to remove with DNA pol I.

ligase

this enzyme catalyzes the formation of phosphodiester bonds between Okazaki fragments forming a continous strand after primers have been replaced.

Polymerase chain reaction (PCR)

used to amplify small fragments of DNA: essentially DNA replication inside a test tube

Taq polymerase

a DNA polymerase enzyme that is heat stable, originally found in prokaryotes in hot springs

what happens in the denaturation step of PCR?

DNA is heated to 98 degrees celsius to break hydrogen bonds between strands (no need for helicase)

what happens during the annealing step of PCR?

DNA sample cooled to 60 degrees celsius, allowing primers to bind to complementary DNA. no need for primase

what happens during the extension step of PCR?

DNA sample is heated to 78 degrees celsius, Taq polymerase replicates DNA (no need for DNA pol III)

PCR set up

A target DNA sample, free nucleotides, primers for target samples, and Taq polymerase in a test tube

gel electrophoresis

after PCR, […] uses an electrical current to move DNA fragments through a gel, where fragments are separated by size.

restriction enzymes

cut DNA molecules at specific sequences before they travel through a gel

DNA fingerprints

restriction enzymes cut sites into unique patterns of bands when a sample is run through a gel.

SNP role in DNA fingerprints

these are able to change cut sites which causes no enzyme activity at that location, thus changing the band pattern

central dogma

refers to the flow of genetic information (DNA → mRNA → protein)

processes in central dogma

transcription (DNA to mRNA) and translation (mRNA to polypeptide chain)

location of transcription

occurs in nucleus for prokaryotes and cytoplasm in prokaryotes

transcription

producing mRNA from a DNA template. allows for only a portion of a genome to be copied (resource efficiency), as well as protecting DNA in a nucleus.

RNA polymerase

the enzyme that performs transcription (elongating mRNA strand) using DNA as a template. synthesizes mRNA in 5’ →3’ direction

3 phases of transcription

initiation, elongation, termination

promoter

a non-coding region of DNA in front of the gene of interest that begins with the TATA box

TATA box

“start line” of promoter, beginning of initiation process

transcription factors

proteins that recognize the TATA box and bind to the promoter

what happens in the initiation phase of transcription?

transcription factors recruit RNA polymerase to the promoter, which begins to temporarily unzip a small portion of the double helix to expose the bases

what happens in the elongation phase of transcription?

RNA polymerase reads the template strand of DNA to synthesize mRNA. as mRNA is synthesized, the RNA nucleotides will temporarily form hydrogen bonds with the template strand. the growing mRNA strand then exits RNA polymerase and the DNA re-zips

template/antisense strand

the strand of DNA that RNA polymerase reads in the elongation phase of transcription

coding/sense strand

the complementary DNA strand of the template strand in the elongation phase of transcription

what happens during the termination phase of transcription?

a termination sequence at the end of the gene is reached, signals for RNA polymerase to release the mRNA and detach from the DNA, concluding transcription

enhancers

a non-coding region of DNA that increases the rate of transcription

silencers

a non-coding region of DNA that decreases rate of transcription

3 functions of non-coding regions of DNA

telomers, genes fro rRNA and tRNA, introns

telomers

repetitive sequences at the end of eukaryotic chromosomes, protect their ends

genes for rRNA and tRNA

RNA is synthesized from these genres, but they don’t code for proteins

introns

base sequences that are removed from the mRNA after transcription

3 post translational modifications

converts pre-mRNA into mature mRNA, mRNA splicing, addition of 5’ cap and poly-A tail

5’ cap

a modified nucleotide added to the 5’ end of mRNA, helps with ribosome bonding during translation, also aids in the export of mature mRNA from nucleus and protects from degradation

poly-A tail

a string of adenines attached to the 3’ end of mRNA, aids in the export of mature mRNA from nucleus and protects from degradation

exons

expressed base sequences (coding regions within a gene)

introns

base sequences that are removed before translation

mRNA splicing

a process where introns are removed from mRNA and stay in the nucleus

snRNPS

catalyze splicing and bind to either side of introns and assemble into spliceosomes

spliceosomes

snRNPs attached to introns that remove them and ligate exons together

alternative splicing

different introns are removed → creates unique mature RNA →unique polypeptides. allows for one gene to provide instructions for several polypeptides

monomer for proteins

amino acids (20 different types)

polymer for proteins

polypeptides

covalent bonds for proteins

peptide bonds

primary level of protein structure

polypeptide chain

secondary level of protein structure

alpha helices and beta pleated sheets

tertiary level for protein structure

3D structure determined by side chains

quarternary level for protein structure

2+ polypeptide chains interacting

protein synthesis in prokaryotes

translation occurs immediately after transcription, therefore is faster than eukaryotes.

genetic code

how mRNA is “decoded” into amino acids

codon

base that reads mRNA in triplets when decoding mRNA into amino acids. each […] codes for a specific amino acid

4 special codons

start codon: AUG/ stop codons: UGA, UAA, UAG

universal characteristic for genetic code

nearly every organism on Earth uses the same genetic code. evidence for LUCA, basis of several biotech techniques

redundant characteristic for genetic code

some amino acids can be coded for by more than one codon

unambiguous characteristic for genetic code

no codon specifies more than one amino acid

genetically modified organisms (GMOs)

a biotechnology technique in which a gene of interest is spliced into another organism, allowing for protein synthesis of the gene by the new organism

uses for GMOs

insulin gene in bacteria (allows for mass production of insulin), pesticide gene in crops (allows for crops to produce protein(s) that act as pesticides so bugs don’t eat them).

1st step of genetic modification process

isolate gene of interest (ex human insulin gene) and amplify gene using polymerase chain reaction

2nd step of genetic modification process

isolate bacterial plasmid and amplify using PCR

3rd step of genetic modification process

use a restriction enzyme to cut the gene of interest and plasmid. insert gene into plasmid, creating a recombinant plasmid

4th step of genetic modification process

insert recombinant plasmid into host organism, creates GMO

5th step of genetic modification process (optional)

allow gene to grow in culture, then extract protein (used for insulin not crops

mRNA

messenger RNA, used to take message from DNA and brings it to ribosome

tRNA

transfer RNA, used to carry amino acids to ribosomes

rRNA

ribosomal RNA, structural component of ribosomes

structure of tRNA

single strand of RNA folded into a 3D structure, bottom contains an anticodon and top binds to amino acid bonding site

anticodon

3 bases of tRNA that will bind to mRNA codon

aminoacyl-tRNA synthase

enzyme that attaches to correct amino acid to the tRNA

1st process of tRNA

aminoacyl-tRNA-synthase attaches to correct amino acid to the tRNA

2nd process of tRNA

brings amino acid to the ribosome and binds to the mRNA codon

structure of ribosomes

made of tRNA and proteins, has a small subunit (mRNA attaches to this) and a large subunit (has 3 binding sites for tRNA: A, P, E sites)

Aminoacyl-tRNA binding site (A site)

site on the large subunit of a ribosome for incoming tRNA with the next amino acid

Peptidyl-tRNA binding site (P site)

site on the large subunit of the ribosome for the tRNA holding the growing polypeptide chain

Exit site (E site)

site on the large subunit of the ribosome for the discharged/empty tRNA to leave ribosome

2 major functions of a ribosome

the binding of the mRNA codon with the tRNA codon using complementary base pairs AND forming a peptide bond between incoming amino acids and the growing peptide chain

1st step of translation initiation

the 5’ end of the mRNA binds to the small ribosomal unit

2nd step of translation initiation

small subunit moves from the 5’ end to the 3’ end and scans for the start codon (AUG)

3rd step of translation initiation

at the start codon (AUG), the initiator tRNA binds to the start codon

4th step of translation initiation

the large ribosomal subunit assembles, placing initiator tRNA into the P-site