Unit 6: DNA structure and replication

transformation

organism takes in DNA (naked DNA) from its environment changing its genotype/phenotype

nucleotide

1. phosphate

2. sugar (deoxyribose)

3. nitrogen base (1 of 4)

rungs

nitrogen bases held together with H bonds

DNA is antiparallel

two DNA strands run in opposite directions, with one strand oriented 5' to 3' and the other 3' to 5'

helicase

separates DNA double helix and unwinds for replication to occur

primase

lays down RNA primers

DNA polymerase I

replaces RNA primer with DNA

DNA polymerase III

adds DNA nucleotide to 3’ end of growing strand

ligase

ligase joins together okazaki fragments

topoisomerase

relieves strain/hypercoiling of DNA by cutting, untwisting, and reattaching

difference between leading and lagging strand

leading: towards replication fork, continuous

lagging: away from replication fork, in chunks, okazaki fragments

semiconservative

when DNA replicates, 2 new strands form containing 1 side of original DNA and 1 side of nucleotides

why do leading and lagging strand get replicated in a different way?

DNA Pol. III can only add nucleotides to 3’ end of forming strand

how does leading strand copy parent strand?

1. helicase opens DNA

2. primase adds RNA primer

3. DNA Pol. III adds bases to 3’ of new strand following fork

how does lagging strand copy parent strand?

1. helicase opens double helix

2. primase adds RNA primer

3. DNA Pol III adds nucleotides to 3’ end and travel away from fork

4. as fork opens, new primer is laid down and new DNA strand begins

5. when okazaki fragments abutt, DNA Pol. I replaces RNA primer w/ DNA

6. Ligase joins DNA strands together

what occurs when mutation happens?

mutations to gametes or the cells that create gametes can be passed to offspring

telomeres

chunks of non coding DNA at the end of chromosomes that arent copied by the lagging strand during replication

why dont germs run out of telomeres?

they have a special enzyme called telomerase that adds telomeres back to DNA

genetic engineering

manipulation of organisms genes/DNA for practical purposes

restriction enzymes

enzymes that look for specific DNA sequenced and cut the DNA at those points (restriction site)

restriction site

• protects organisms from foreign/viral DNA

• area cut by restrictions enzyme and leaves sticky ends

restriction fragment

DNA and restriction enzymes are mixed and DNA is cut at every restriction site

plasmids

asexual, small circular DNA molecules that replicate independent if the organismc genome

recombinant DNA

cutting and pasting DNA using enzymes

• introduced new genes into organisms DNA

• plasmids can be taken in by other bacteria by transformation

whats recombinant DNA used for?

1. bacterial plasmid is removed and cut with restriction enzyme

2. a human gene (insulin) is cut out of a sample of human DNA using restriction enzymes

3. plasmid and human gene combine and join using ligase

4. recombinant plasmid reinserted into bacteria

5. bacteria and all of its offspring use the human gene to make insulin

gel electrophoresis

technique used to separate DNA or other molecules based on sized

• DNA is negative so restriction fragments move towards positive end

• shorter segments travel further in the gel

• similar DNA will have similar bands

PCR (polymerase chain reaction)

method used to clone DNA fragments and uses Taq polymerase. also used to produce copies of a particular DNA sequence

Taq polymerase

heat stable DNA polymerase

why is it important to use Taq polymerase?

allows to heat up several times without denaturing

PCR steps

1. DNA heated and causes DNA strand to separate

2. DNA is allowed to cool, allows DNA primers to attach to opposite ends of target region (TAQ)

3. DNA polymerase adds nucleotides to 3’ end until entire target sequence copied

4. repeat many times

gene editing

CAS-9 : bacterial protein that is a restriction enzyme designed to destroy viral DNA

CRISPR : region of DNA used to create an RNA “guide” that telld CAS-9 where to cut DNA

what is gene editing used for?

1. knock out genes → CRISPR/CAS-9 targets a gene of interest → gene is cut → no proper DNA provided → random nucleotides repair “cut” and gene no longer functional

2. alter existing genes/ promote function → CRISPR/CAS-9 cuts defective gene → DNA template of functional gene provided → nucleotides are replaced to make gene functional

what is the role of DNA in organisms?

1. hereditary material

2. tells cells how to make proteins

transcription

when a gene in DNA is used to construct an mRNA molecule

why is transcription important?

1. The presence of specific transcription factors determined which genes will be transcribed

2. RNA Pol III binds to DNA and separates double helix, beginning to construct an mRNA molecule

How is mRNA different from DNA?

1. DNA is double stranded and mRNA is single stranded

2. mRNA can leave the nucleus but DNA cant

3. mRNA has uracil instead of thymine

4. Different sugars

What is the role of the TATA Box/ promoter in transcription?

It is where RNA polymerase binds to start transcription

• RNA polymerase only binds to TATA when appropriate transcription factors are present

What do transcription factors do?

They dictate where RNA Pol III will attach to the DNA

• they control what genes are transcribed and turned into proteins

• Changing transcription factors (number and types), cells can respond to changes and change the proteins they produce

Transcription initiation complex

RNA polymerase and transcription factors bound to the promoter, allowing transcription to begin

How do eukaryotic organisms terminate transcription at the end of a gene?

Due to polyadenylation signal (AA UAAAA)

• when RNA polymerase reaches this sequence, mRNA unattaches and transcription stops

What happens to mRNA before it exits the nucleus?

mRNA processing:

1. Introns cut out, exons are reattached

2. Poly A Tail and 5’ cap are added

   • These protect mRNA from hydrolytic enzymes in cytoplasm

RNA Splicing

Removing introns/attach extrons (does not include prokaryotic)

Alternative splicing

Cells can have 1 pre mRNA to make multiple different proteins.

• cells can change the regions considered exons/introns to make different final transcripts/proteins

Spliceosome

Large complex that processes pre mRNA and removes introns, joins together exons

Codon

Group of 3 mRNA bases coding for a specific amino acid

Anti codon

Series of 3 bases on tRNA molecule

tRNA

Transfers RNA and carries correct amino acids into position. Has anticodon that base pair with mRNA codon

What happens to mRNA at the ribosome?

1. Groups of 3 codons attract a tRNA molecule with a compatible anticodon.

2. tRNA carries specific amino acid which is brought into proper position to be joined to an existing polypeptide

3. New amino acid joined to the polypeptide by a peptide bond

4. Ribosome moves to next codon, repeat

How do tRNA pick up amino acids?

Enzyme (amino actyl synthetase) attaches correct amino acids to a tRNA

Where does translation start?

Codon AUG

How does translation end?

When a stop codon is reached, it codes for a release factor and breaks bond between tRNA and polypeptide. Polypeptide is released in cytoplasm

wobble

Flexibility in base pairing of the 3rd anticodon base with mRNA codon

Point

Change in 1 DNA nucleotide

Insertion

Adding 1 base/nucleotide (results in frameshift)

Deletion

removing 1 base/nucleotide (results in frameshift)

Silent mutation

Point mutation does not cause a different amino acid to be used (no effect)

Missense

Wrong amino acid used due to mutation

Nonsense

Mutation causing a stop codon

Mutagens

Chemical causing mutations

Ribosome structure ns function

Made out of: tRNA & proteins

Used in translation: mRNA codons attract tRNA carrying amino acids used to build a protein