BIOL 200 Midterm Review

Central Dogma

DNA > RNA > Protein

Nucleotides

base, sugar, phosphate

Glycosidic Bond

Base and sugar bond

Nucleotide monomers

phosphates = acidic characteristics, covalently bonded to suger (phosphodiester bond)

Hydrogenous base = covalently bond to sugar

Sugar

Purines

2 ring base, Adenine, Guanine

Pyramidines

1 ring base, RNA = uracil, cytosine, DNA = thymine, cytosine

Nucleoside

base, sugar

Amino Acid

building blocks of polypeptides, determines the shape of the polypeptide, 20 types

Protein functions

catlysis, transport, signalling, structure, motor, regulatory (control protein activity/gene function)

Amino Acid Structure

Amino end (N-terminus), Carboxyl end (C-terminus), R groups

Special a.a.

Cysteine, glycine, proline

Cysteine

special a.a with sulfhydryl group, form disulfide bonds

Glycine

special a.a. with H symmetrical, intra/inter cross linking

Proline

special a.a. rigid ring structure

DNA shape

5' end: free phosphate group attached to sugar phosphodiester bond: links nucleotides

3' end: free hydroxyl group on terminal end sugar

DNA "backbone"

repeating phosphate-pentose units, holds no info

outside of dbl bond

within DNA structure

3D structure of DNA

watson and crick, 2 polynucleotide strands wound together to form dbl helix (anti //), bases stacked

Base pairing

complementary base

T dbl bonds to A, C triple bonds to G (stronger = more H bonds)

Right-handed Helix DNA

most DNA in cells

B DNA

major and minor groove, important for DNA-protein interactions, ex: TBP

R-H helix

A DNA

low humidity or high [salt], B DNA turns into A DNA

RNA-DNA, RNA-RNA exist in this form

R-H helix

- smaller, wider

Z DNA

short DNA molecule of alternating purines and pyramidines

formed after transcription, tag for transcribed genes

L-H helix

- taller, longer

DNA denaturation and renaturation

important for DNA replication/transcription

unzipping and re-annealing of DNA

RNA secondary structures

hairpin, stem-loop

RNA tertiary structure

pseudoknot

Primary structure of protein

a.a. sequence

amino end, amide groups, r groups, carboxyl ends

Peptide Bond

linkange of one a.a. to another, on C terminus linking to next N terminus

Residue

amino acid

Secondary structure of protein

local folding

alpha-helix: stabilized by H bonds

beta sheet: 5-8 residues, r groups away/toward you, laterally packed beta strands, // or anti //

Beta turn: most common, 3-4 residues, glycine/proline twists and turns

Tertiary structure of protein

long range folding

stabilized by hydrophobic interactions b/w non-polar side chains

H bonds b/w polar side chains

Disulfide bonds b/w cysteine residues (covalent bond)

Quaternary structure of protein

multi-meric structure

pores (4 proteins) = potassium ion channel protein

Supramolecule structure of protein

large-scale assembly

10-100 polypeptides chains

general transcription factors: RNA polymerase, mediator complex, promotor, pre-initiation transcription complex

Motifs

combos of secondary structure proteins

Coil-Coil Motif

hydrophobic interactions

fibrous proteins

ex: collagen

Helix-loop-helix Motif

ionic bonds involving Ca2+

ex: Ca2+ binding proteins

Zinc-finger Motif

contains Zn2+

ex: RNA, DNA, binding proteins

Domains

need these for certain proteins to function

ex: pyruvate kinase needs 3 domains

Reshuffling of motifs and domains

new proteins made

Ingredients for transcription in bacteria

DNA template, ribonucleotides (monomers for RNA polymerization), RNA polymerase (catalyze synthesis of RNA

Ist step of transcription

Initiation

- polymerase binds to promoter (upstream of gene) in duplex DNA

forms "closed complex"

- polymerase melts duplex DNA

forms "open complex"

- polymerase catalyzes phosphodiester linkage of two initial rNTPs

rNTP

ribonucleotide tri-phosphate, building blocks of RNA synthesis

N = G, C, A, U

2nd step of transcription

Elongation

- polymerase goes from 3' to 5'

- continues to melt DNA

- adds rNTPS to growing RNA

- formation of phosphodiester bonds b/w 3' OH and alpha phosphate group of incoming rNTP

3rd step of transcription

Termination

- at stop site, polymerase releases completed RNA and dissociates from DNA

Holoenzyme

subunit of RNA polymerase

consists of core enzyme + sigma factor

alpha: loading entire onto transcript

beta: helps with phosphodiester linkage

Sigma factor

subunit of RNA polymerase

scans DNA until encounters promoter to bind with and form "closed complex"/to start transcription

How does sigma factor recognize promoter region?

RNA polymerase subunit

binds to specific sequence motifs (-10, -35 regions = start, stop site)

Messenger RNA

mRNA

genetic info from DNA in form of codons

Transfer RNA

tRNA

key to decipher codons in mRNA

each tRNA has an associated a.a. and anticodon

Ribosomal RNA

rRNA

assiciates with proteins to from ribosomes

tRNA structure

folds with itself

acceptor stem, loop, anticodon, loop

specific tRNA to a.a.

Start and stop codon

Met = start

codon AUG

3 Stop codons

Wobble base pairs

Guanine-Uridine

Adenosine-Inosine

Cytidine-Inosine

Uridine-Inosine

Where does wobble base occur?

b/w the 3rd position of a codon and 1st position of anticodon

ex: anticodon GAC (3,2,1) can b.p. with codon GUC (3,2,1) or GUU (3,2,1)

1st step of protein synthesis

Initiation

- initiation factors associate w/ 30s subunit = pre-initiation complex (IF1 and IF3)

- IF1 and IF2-GTP loads 50s subunit and forms 70s initiation complex

IF1 and IF3

Initiation factors in protein synthesis

loads mRNA and initiates aminoacyl-tRNA forming 30s initiation complex

30s initiation complex

In initiation step of protein synthesis

binds the transcript at initiation codon AUG

2nd step of protein synthesis

Elongation

- elongation factors (EFs) = required for addition of a.a. (ribozyme, 23s RNA)

- translocation occurs

Ribozyme, 23s RNA

Required for elongation of protein synthesis

carries out peptdyltransferase rxn = makes peptide bond

Translocation

tRNA movement

anticodon of tRNA moves to the next codon of mRNA

P site of tRNA displaced

3rd step of protein synthesis

Termination

mRNA-ribosome-tRNA-peptidyl complex reaches stop codon

release factors mediate termination

RF1 and RF2 = mimic tRNAs

RF3-GTP: catalyzes the cleavage of the peptidyl-tRNA, releases protein chain

ribosome recycled

Parental strand

template for the formation of a new daughter strand

Daughter strand

complementary to parental

Semi-conservative mechanism

DNA replication proceeds through this system

dbl stranded DNA splits up, and each strand acts as parent strand

each parent strand makes a complementary strand = daughter strand

Nucleotide (DNA) polymerization

DNA polymerase: catalyzes the polymerization

Substrate = 5' deoxynucleoside-triphosphates (dNTPS, N = A, C, T, G

Primer: can be DNA or RNA

proceeds in 5' to 3'

How are dNTPs added in DNA polymerization?

through the formation of phosphodiester bonds on the 3' hydroxyl of terminal sugar

RNA polymerization does not require these

primers

If primer of DNA polymerization is RNA?

addition of dNTPs by DNA polymerase will result in molecule that is RNA at 5' end and DNA at 3' end

DNA helicase

unwinds DNA duplex so DNA can be replicated

part of origins of replication

Duplex DNA

initiates unwinding at specific regions

part of origins of replication

Origins of replication

where unwinding is initiated

tends to be AT-rich

prokaryotes can have one

eukaryotes can have 1000s...

Primase

specialized RNA polymerase

forms short RNA molecule complementary to a single-stranded region of the unwound duplex DNA

DNA polymerase

extends primer in DNA replication

eventually forms the new daughter duplex DNA

DNA replication direction

5' to 3'

two strands of duplex DNA are anti //

replication proceeds bidirectionally

Leading and lagging strand in DNA synthesis

Leading strand: continuous, replicated in 5' to 3' by DNA polymerase, follows movement of replication fork

Lagging strand: discontinuous, formed in the opposite direction of movement of replication fork

Lagging strand components

Short RNA primer: DNA is elongated from RNA primer (made by primase) by DNA polymerase

Okazaki Fragment: short discontinuous fragments containing RNA and DNA

DNA ligase: ligates/sticks RNA components of Okazaki fragments with DNA, heals break in sugar-phosphate DNA

SV40

Perfect system to study DNA replication

virus that infects monkeys and humans

RPA

used in leading strand synthesis

Replication Protein A

- binds single-stranded DNA

- keeps single-stranded DNA template in optimal conformation for DNA polymerase (ssDNA can fold with itself)

Pol ε

used in leading strand synthesis

-DNA polymerase that replaces pol α and takes over synthesis

Rfc

used in leading/lagging strand synthesis

- replication factor

PCNA

used in leading/lagging strand synthesis

- Proliferating Cell Nuclear Antigen

- homotrimetric protein

- replaces RPA as synthesis is carried out

- prevents complex (pol ε/Rfc/PCNA) from disassociating from template

Primase/Pol α complex

used in leading and lagging strand synthesis

- primase forms RNA component of primer

- pol α (DNA polymerase) extends primer with DNA

Pol ε/ Rfc/PCNA complex

used in leading strand synthesis

- replaces primer sequence with DNA

- extends primer sequence and continues synthesis

Pol ∂/Rfc/PCNA complex

used in lagging strand synthesis

- replaces primase/pol α complex

- completes the synthesis of an okazaki fragment

- RNA, DNA and terminates where RNA primer meets 1000 b.p.

Ribonuclease H and FEN-1

In lagging strand synthesis: displaces the RNA component at the 5' ends of Okazaki fragments

DNA replication: removal of primers

Pol ∂

used in lagging strand synthesis

- replaces RNA with DNA and takes over synthesis

Large t-antigen

used in DNA replication

- unwinds the DNA duplex

- acts as helicase, driven by hydrolysis of ATP

Origin recognition complex (ORC)

eukaryotic chromosomal DNA contains multiple of these

- six subunit protein

- binds to origins

- associates w/ other proteins to load helicases (MCM/minichromic maintenance)

Mutations

permanent, transmissible (capable of being transmitted by infection) changes to genetic material of cell

Mutagens

chemical compounds that increase frequency of mutations

ex: UV radiation, ionizing radiation (X-rays/atomic particles)

Carcinogen

an agent that causes cancer

a mutagen

Proof reading by DNA polymerase

1st line of DNA repair

- DNA polymerase induces 1 error every 10 000 incorporated nucleotides

- measured rate of incorrect = 1 every 1 000 000 000

3' to 5' exonuclease activity: chews off single nucleotide from the end of a polynucleotide chain

pol III: extends growing strand after exo chews off error, finger domain closed when correct NTP enters

thumb: once correct NTP binds, thumb wraps around DNA (helps DNA processivity, translocation, position)

eukaryotes: DNA polymerase ∂ has 3' to 5' exonuclease activity

Base excision repair

DNA repair

- removes bases that can cause mutations

DNA glycosylase

involved in 1st step in base excision repair

- hydrolyzes hydrolyzes the covalent bond between a T base and the sugar-phosphate backbone

- recognizes non watson-crick base pair

Apurinic/Apyrimidic endonuclease I (APE I)

involved in 2nd step in base excision repair

- cuts the DNA backbone

Ap lyase

involved in 3rd step in base excision repair

- associated with DNA polymerase B

- removes the deoxyribose phosphate

- removes groups from substrate and leaves a dbl bond

DNA polymerase B and DNA Ligase

involved in 4th step in base excision repair

- GAP REPAIR

- dna polymerase B fills the gap

- dna ligase seals the knick in sugar-phosphate backbone

Mismatch excision repair

DNA repair

- fixes erroneous insertion, deletion and mis-incorporation of bases that can arise during DNA replication and recombination (in newly synthesized strand)

Steps of mismatch excision repair

1. MSH2 and MSH6 bind to daughter strand and triggers MLH1 endonuclease (cuts w/i strand) and PMS2 binding activity

2. DNA helicase unwinds and DNA exonuclease digests the error cut

3. Pol ∂ and DNA ligase: carries out gap repair

Nucleotide excision repair

mutagen modifies bases that distort the normal shape of DNA

- cuts out nucleotide causing error/dimers that are bad for us

ex: UV on our DNA = thymine-thymine dimer residue

Steps of nucleotide excision repair

1. XP-C: recognizes the problem

2. TFIIH: has helicase function

3. XP-F and XP-G: endonucleases

4. DNA polymerase and ligase: GAP repair