Biochemistry Class 3 - Molecular Biology

What is the monomer for nucleic acids?

Nucleotides and nucleosides

What are nucleotides? Nucleosides?

Nucleotides: sugar, base, phosphate

Nucleoside: sugar and base

What is ribose? Structure

See image

What is deoxyribose? Structure

See image

What are building blocks of DNA?

2’ deoxynucleoside triphosphate

dNTP

How are dNTPs connected?

3’ O on first dNTP connects to 5’ phosphate of second

Two phosphates leaving second dNTP releases necessary energy

What direction are nucleic acids synthesized? Direction they are written in?

5’ to 3’

How is DNA structured?

5’ to 3’ synthesis

Antiparallel

Complementary bases paired

Bases held together via phosphodiester bond

What are pyrimidines?

Cytosine and Thymine and Uracil

Six membered ring

What are purines?

Adenine and Guanine

Six membered ring stuck to five membered ring

How are A, C, T, G held to their complementary bases?

C and G by three H bonds (more stable)

A and T by two H bonds (less stable)

How do prokaryotic and eukaryotic genomes differ?

Prokaryote: single circular genome, in cytoplasm

Eukaryote: chromosome strings, in nucleus

How do prokaryotes protect themselves from restriction enzymes?

Restriction enzymes: chop up DNA to restrict virus growth

Methylation of DNA prevents REs from binding its own DNA

Supercoil DNA

What does DNA gyrase do?

Supercoils prokaryotic DNA

How is DNA packaged in eukaryotes?

DNA → wrap twice around histone → nucleosome → wind into chromatin

What is a centromere?

Only in eukaryotic genomes

Where spindle fibers attach

Where sister chromatids attach

What is the short arm of a chromosome? Long?

Short: p arm

Long: q arm

What is a telomere?

Only on eukaryotic genomes

Ends of linear chromosomes (made of single and double stranded DNA)

Stabilize end of chromosome by capping it

Consist of short repeats (As and Ts)

What is the central dogma?

DNA → RNA → proteins

Use RNA as an intermediate so you do not damage DNA by working with it directly

What is transcription?

DNA → RNA

Both use nucleotides

What is translation?

RNA → protein

Nucleotides → amino acids

What is the start codon?

AUG

Specify methionine

What are the stop codons?

UAA

UGA

UAG

Do not code for amino acids

How many codons are there?

61

What is the tryptophan codon?

UGG

What are sources of mutations?

Polymerase errors, endogenous damage, exogenous damage, transposons

How do polymerase errors affect DNA?

Cause point mutations, small repeats, or small insertions/deletions

What are point mutations?

Wrong nucleotide put in

What are small repeats?

Polymerase slips off DNA → reputs itself in almost the same place → re-replicates a few bases

What are insertions/deletions?

Insertions: caused by chemicals that trick polymerase into thinking it should put a base somewhere it should not

Deletions: damage causes bases to polymerize in a way that makes them unrecognizable, so polymerase thinks there are none there

What are point mutations? Types and effects

Single base pair change

Missense → changes the amino acid sequence

Nonsense → changes a codon into a stop codon

Silent → no change in amino acid sequence

What are frameshift mutations? Types and effects

Insertions and deletions that change the reading frame

How do exogenous and endogenous damage affect DNA? What causes this?

Endogenous: ROS, physical damage → oxidized DNA, crosslinked bases, physical damage, can lead to polymerase errors

Exogenous: radiation, chemicals → pyrimidine dimers, double strand breaks, translocations, physical damage, intercalation, polymerase errors

What damage do UV and X-ray radiation cause to DNA?

UV: pyrimidine dimers

X-rays: double strand breaks, translocations

What are transposons and how do they affect DNA?

Genes that move around in the DNA

Large insertions/deletions, inversions, duplications

What is the anatomy of a transposon

Transposase enzyme flanked by DNA

Transposase cuts and pastes DNA into other places of DNA

DNA flanking transposons has inverted repeats

What is an IS element?

Transposase flanked by inverted repeats

What is a complex transposon?

Transposase and gene(s) flanked by inverted repeats

What is a composite transposon?

Two sets of transposons with inverted repeats flanking them

Central region between them

How are transposons inserted?

1. Transposase is made

2. Transposase cuts transposon out

3. Transposase puts the transposon elsewhere

What are effects of composite transposons that run in the same direction?

Deletions

Chromosomal rearrangement (same or other)

What are effects of composite transposons that run in the opposite direction?

Inversions

Chromosomal rearrangement on the same chromosome

How are polymerase errors repaired?

Mismatch repair pathway (after replication)

Nucleotide excision repair (before replication)

How are transposon errors repaired?

They generally are not

How does mismatch repair work?

Repairs polymerase errors after replication

Recognizes unmethylated DNA as the replicated (messed up) strand

Uses old strand as template

How does nucleotide excision repair work?

Prior to replication

Removes and replaces the bad base

How are endogenous and exogenous DNA damage repaired?

Both: nucleotide excision repair, end joining (homologous and non)

Exogenous only: direct reversal

How does homologous end joining work?

Repairs double stranded DNA breaks

Uses sister chromatid as template

After replication, as it requires sister chromatid

How does nonhomologous end joining work?

Repairs double strand DNA breaks

No sister chromatid for template

Sticks shit together → mutagenic, but better than broken

What kinds of errors are caused by nonhomologous end joining?

Translocations

What is direct reversal?

Visible light reverses dimerization caused by UV radiation

What are the rules of DNA replication (4)?

1. Semiconservative

2. 5’ to 3’ direction

3. Requires a primer

4. Requires a template

What does helicase do?

Binds to origin of replication and uncoils DNA

What does topoisomerase do?

Cuts DNA to relax supercoiling caused by helicase

What does primase do?

Puts down the RNA primer

What does DNA polymerase do?

Replicates DNA, proofreads, removes RNA primer

What does ligase do?

Fuses together Okazaki fragments

How is DNA replicated in prokaryotes? AKA Theta replication

1 origin of replication

5 DNA polymerases

What is DNA polymerase III?

In prokaryotes

Main replicating enzyme

5’ to 3’ polymerase and 3’ to 5’ exonuclease activity

Starts 400 bases downstream of the origin

Fast

What is DNA polymerase I?

In prokaryotes

Adds nucleotides at the RNA primer until polymerase III can start

Has 5’ to 3’ polymerase

Has 3’ to 5’ and 5’ to 3’ exonuclease activity

Slow

Helps with DNA excision repair

What does DNA polymerase II do?

5’ to 3’ polymerase and 3’ to 5’ exonuclease

Backup for DNAP III

Does DNA repair

Not necessary for cell to live

What do DNA polymerases IV and V do?

Error prone 5’ to 3’ polymerase activity

DNA repair

Not super important for cell

What do exonucleases do?

Allow errors that were just made to be corrected

Only works at the end of strands, not internally

How does DNA replication in eukaryotes work?

Multiple origins, replication bubbles, several DNA polymerases with complex subunits

What is the end replication problem?

RNA primers add at lagging end, but they can never be put down at the very very end → some cannot be replicated → small single stranded part of DNA (telomere)

Every round of replication shortens the telomere

What is telomerase?

In eukaryotes

Has reverse transcriptase activity and an RNA primer built in

Lengthens telomeres by adding repetitive nucleotide sequences

What is rRNA?

ribosomal RNA

Catalytic part of ribosome

What is mRNA?

Messenger RNA

Sequence of codons determines the amino acid sequence of the protein

What is tRNA?

Transfer RNA

Carries amino acids to ribosomes

What is hnRNA?

Heterogeneous nuclear RNA

Initial unprocessed transcript

What are siRNA and miRNA?

Micro RNA and Small Interfering RNA

Help regulate gene expression

How are replication and transcription similar/different?

Both: start site, 5’ to 3’, DNA template

Replication: no stop site, needs primers, proofreading

Transcription: stop site, no primer, no proofreading

What are template vs coding strands?

Coding: AKA sense, same sequence as RNA

Template: AKA antisense, used to make RNA

How does transcription work?

RNAP binds to promoter → reaches start site → starts transcribing → reaches stop site → polyadenylase adds poly A tail

How is translation primarily regulated? Two types

At the transcription level (once you have mRNA, you basically have proteins)

Via promoters or DNA binding proteins

How do promoters regulate translation?

Strong promoters have high affinity for RNAP → lots of transcription

Weak ones have low affinity for RNAP → less transcription

How do DNA binding proteins regulate translation?

Repressors: bind DNA and prevent transcription

Enhancers: bind activators → bind transcription factors in proximity to promoter → increase transcription

How are prokaryotic and eukaryotic transcription different?

Prokaryotic: transcription and translation at same place/time (cytosol); no mRNA processing; polycistronic; one RNAP

Eukaryotic: transcription in nucleus, translation in cytosol; mRNA must be processed; monocistronic; three RNAPs

How is mRNA processed?

In eukaryotes

5’G cap added

3’ poly A tail

Splicing to remove introns

What does it mean to be polycistronic?

From one mRNA, many different proteins can be made

What do RNAP I, II, and III do?

In eukaryotes

RNAP I: transcribes rRNA

RNAP II: transcribes mRNA

RNAP III: transcribes tRNA

What is the anatomy of a tRNA?

Amino acid attaches at 3’ end

Anticodon at second loop

What is wobble pairing?

Third position anticodon has more flexible base pairing than the others

Reduces how many codons you need to make

G in tRNA can bind to U or C on mRNA

U in tRNA can bind to A or G in mRNA

A always binds U

C always binds G

What is inosine?

Adenine tRNA can convert into inosine

It can bind to A, U, or C

How are tRNAs made?

Aminoacyl tRNA synthetase loads amino acid on the tRNA

Costs 2 ATP per amino acid

How do prokaryotic and eukaryotic ribosomes differ?

Prokaryotic: 50S large subunit, 20S small subunit; total 70S

Eukaryotic: 60S large subunit, 40S small subunit; total 80S

S is a measure of how fast you sink in a centrifuge (aka size)

How do ribosomes work?

Growing protein held on P site

Amino acids added at the A site

mRNA binds to ribosome large subunit (first codon in P site, second in A) → tRNA binds to P-site → tRNA binds to A site → break bond between tRNA and amino acid at P site (release 2 ATP) → translocation down one codon → → reach stop codon (no tRNA for this) → bind release factor → breaks final tRNA - amino acid bond

ATP released is used to make peptide bonds

How much ATP is used at each step of making a protein?

tRNA loading: 2 per amino acid

Initiation: 1 per peptide

A site binding: 1 (per amino acid minus 1)

Translocation: 1 (per amino acid minus 1)

Termination: 1 per peptide

ATP used = # of amino acids x 4

How are proteins post-translationally modified?

1. Folded by chaperones

2. Can add covalent modifications (disulfide bridges, glycosylation, phosphorylation)

3. Processing (ex: removal of some parts to activate it)

How is insulin post-translationally processed?

Folded → disulfide bridges → cleavage into insulin and C peptide (non functional but can be measured)