Bio 102 exam 3 (DNA structure + function)

DNA is made up of

Polymer of nucleotides

• 5-carbon ribose

• Base on 1’ carbon

• Phosphate group on 5’ carbon

Why can DNA be replicated?

Because of its structure:

• Double stranded— each strand has all info needed to make new identical DNA molecule

• Strands complementary

• Semi-conservative

Semi-conservative DNA replication

Each strand of the original double helix serves as a template for the synthesis of a complementary strand; each daughter DNA molecule thus contains one parental strand and one new strand

Origin of replication

First spot where hydrogen bonds are broken between nucleic acid strands for DNA replication

Prokaryote replication

Prokaryotes have small circular genomes

• Single origin of replication sufficient to copy whole genome

• New DNA made in both directions, on both strands, at origin

Eukaryote replication

Eukaryote genomes have many origins of replication

• Longer genomes than prokaryotes

• Eukaryotic DNA polymerases slower than prokaryotic DNA polymerases

Helicase

Breaks H bonds to separate DNA strands

DNA polymerase

• Builds new strand of DNA

  • DNA made 5’ to 3’, so DNA polymerase travels 3’ to 5’ along template strand

Single stranded binding proteins

Make sure strands don’t stick back together after helicase unzips DNA at replication fork

Topoisomerase

Untangles DNA helix as strands pulled apart

Primase

• Creates RNA primer

  • Starter set of RNA nucleotides that provide free 3’ OH upon which new DNA nucleotides can be added

DNA is built in the _ direction

5’ to 3’

Leading strand

Strand where DNA polymerase travels toward replication fork

Lagging strand

Strand where DNA polymerase travels away from replication fork

Okazaki fragments

Ends contain specialized polymerase that removes and replaces RNA primer from adjacent Okazaki fragment

DNA ligase

Joins Okazaki fragments by catalyzing formation of covalent bond

Polymerase Chain Reaction

• PCR

• Test tube version of DNA replication

PCR steps

1. Denaturation: separation of DNA strands with heat

2. Annealing: allow primers to bind to DNA

3. Extension: use a DNA polymerase to synthesize new DNA

PCR uses _ to build new DNA

DNA polymerase

• Primers pre-made to target one spot in genome

  • Made of DNA, not RNA

• dNTPs must also be added

DNA polymerase binds to _ end of primer

3’ 

• Starts extending new DNA strand

After the second round of PCR, new DNA is what length?

• Exact length of the two primers and all the DNA between them

  • After first round of PCR, the new DNA extends past the other primer

In gel electrophoresis, DNA moves towards

• Positive end

• Smaller fragments move faster

If n= number of PCR cycles, _ = the number of DNA molecules that will be provided

2 × 2^n

Transcription

• Template strand of DNA used to make mRNA

• mRNA then translated by a ribosome to make a polypeptide (protein)

mRNA built _ direction

• 5’ to 3’

• RNA polymerase moves 3’ to 5’

Promoter

• Non-transcribed sequence of DNA, just before the start of transcription

• Positions RNA polymerase so it’s in the right spot to start transcribing

Upstream vs downstream

• Downstream: direction RNA polymerase moves (3’ to 5’)

• Upstream: where promoter lies

Prokaryotic promoters

• Critical DNA sequences centered at -35 and -10 bp before the transcription start site

• Sigma factor bind to -35 and -10 regions

  • Core RNA polymerase assembles on top of sigma factor

Eukaryotic promoters

• Critical promoter DNA sequence called the TATA box

• Number of transcription factor proteins assemble at the promoter, then RNA polymerase binds

Transcription —> translation in prokaryotes vs eukaryotes

• Prokaryotes: Translation begins immediately after transcription

  • Bacteria: co-transcriptional translation where translation starts while transcription still happening

• Eukaryotes: RNA processed after transcription, then moves to translation

Steps of eukaryotic RNA processing

1. Capping 5’ end

2. Tailing 3’ end (poly-A tail)

3. Splicing out introns

5’ cap and poly A tail purpose

Protect mRNA from exonuclease enzymes digesting nucleic acids

• Added post-transcriptionally

Introns

• Intervening sequences, must be removed before translation

• Spliceosome brings two exons together and cuts out intervening intron

Translation

• Production of a polypeptide based on info provided by mRNA

• Accomplished by ribosomes

tRNA

brings amino acids into the ribosome, adding it to the growing polypeptide chain

Amino acids attach to the _ end of tRNA

3’

Secondary and tertiary tRNA structure stabilized by

• Hydrogen bonds

• tRNA base pairs with itself

Codon vs anticodon

• Codon: sequence of 3 nucleotides on mRNA that specifies a particular amino acid

• Anti-codon: complementary sequence of 3 nucleotides on tRNA that pairs with codon

Translation start/stop

• Starts when ribosome encounters AUG start codon (methionine/M) in mRNA

• Stops when ribosome encounters one of three stop codons in mRNA

Genetic code is:

• Redundant

  • Most amino acids can be specified by multiple different codons

• Universal

  • Bacteria can translate human mRNA and vice versa

• Unambiguous

  • Each codon is read as only one amino acid

Ribosome has space for _ tRNA molecules at a time

• 3

• E P A sites

• Each tRNA molecule enters on right, moves to middle, then moves to left, then exits ribosome

How does translation begin?

• Small subunit on ribosome assembles on 5’ UTR of mRNA

• First tRNA then base pairs with the start codon

• Large subunit joins

  • First tRNA in P site, carrying methionine

How are amino acids added to chain in translation?

• Once translation is initiated and first tRNA is in P site, a second tRNA enters the A site, carrying the next amino acid

• Amino acid in A site covalently bonds with polypeptide chain in P site

• Both tRNA molecules move forward a spot

• P-site tRNA now in E site, has no amino acid so leaves

What happens when ribosome reaches a stop codon?

1. A-site accepts release factor

2. Release factor promotes hydrolysis, freeing the polypeptide

3. Ribosome subunits and other components dissociate

N-terminus and C-terminus of polypeptide chain

• N-terminus = “front” end

• C-terminus = “back” end, to which a new amino acid can be added

Mismatch repair

Method of DNA repair

Steps of mismatch repair

Scanning enzymes detect the shape of a mismatched base pair, then

1. Endonuclease enzyme cuts sugar-phosphate backbone of newly made DNA

2. Exonuclease enzyme chews away mismatched pair and surrounding DNA

3. DNA polymerase fills gap, adding first nucleotide on free 3’ OH

4. Ligase seals gap, as free 3’ OH of last newly made base not connected to 5’ phosphate of next base

Mutations in DNA can occur in

• Protein-coding regions of gene

• Regulatory regions of gene

• Stretches of DNA between genes

Mutations most to least likely to dramatically alter gene function

Promoter and coding regions > UTR > intergenic regions

Synonymous mutation

Mutation doesn’t change amino acid code

Nonsense

Instead of an amino acid, mutation creates stop codon

Missense

Mutation changes the coded amino acid to a new amino acid sequence

Synonymous, nonsense, and missense mutations are examples of

Single Nucleotide Polymorphisms (SNPs)

Indel mutations

• Insertions and deletions

• Indel that is a multiple of three will add/subtract a whole number of amino acids

• Indel that isn’t a multiple of three causes a frameshift

Frameshift

Ribosome reading frame for translation is altered and amino acids will be provided incorrectly from that point on