adenine

1. Draw structure

2. Where is found?

3. pKa of N-1

cytosine

1. Draw structure

2. Where is found?

3. pKa of N-3

Draw/define the two furanose ring conformers (endo and exo).

Which is more stable?

[image]

C2'-endo is most common, found in B-form DNA

C3'-endo is common in RNA

guanine

1. Draw structure

2. Where is found?

3. pKa of N-1

thymine

1. Draw structure

2. Where is found?

3. pKa of N3

In a five-membered sugar, what are the distances between the two phosphate groups?

A. C3'-endo

B. C2'-endo

A. For C3'-endo, it is 5.9 Å

B. For C2'-endo, it is 7.0 Å

Alpha sugars have -OH next to the ether O down.

Beta sugars have -OH next to ether O up.

mutation in which a part of the DNA is missing. anywhere from 1 base pair to parts of chromosomes. 

5’--------GAATTC---------3’

3’--------CTTAAG---------5’

Deletion:

5’--------GAATC----------3’

3’--------CTTAG----------5’

mutation involving insertion of new DNA, ranging from 1 to many base pairs

5’----------GAACTTC---------3’

3’----------CTTGAAG---------5’

Point Mutations

(two types)

mutation involving a change in the nucleotide. 

Transitions: Purine to other purine or pyrimidine to other pyrimidine. 

Transersions: Purine to Pyrimidine or Pyrimidine to Purine.

5’--------GAATATTC--------3’

3’--------CTTATAAG--------5’

changes a codon, but not the encoded amino acid residue

TGT (Cys)--> TGC (Cys)

GCA (Ala)--> GCN (Ala) (N = any)

–  This is possible because the code is degenerate

changes the encoded residue

TGT (Cys)--> TGG (Trp)

an amino acid-encoding codon becomes a stop codon

TGT (Cys)--> TGA (STOP)

NH2- Met-Thr-Leu -Lys –COOH

         5’-ATG-ACC-TTG-AAA-TAA-3’

NH2-Met-Pro –COOH       

5’-ATG-CCT-TGA-AAT-AA-3’

*delete A

-change can be neutral, silent mutation.

-change can have a subtle effect. ex) Lys to Arg    

may happen in complex genetic diseases

-change can have measurable effects. (pronounced reduction in activity)

-change can change protein function. a new substrate might be recognized. 

-change can complete eliminate the proteins ability to function. 

evironmental factors - living results in exposure to different chemicals and damaging effects

i.e. ethanol, smoking, etc

another cause of DNA damage. 

Deupurination or Depyrimidation resulting in abasic site AP  site (apurinic or apyrimidinic site)

-various alkylating agents (methyl, ethyl, or alkyl) damage DNA

-i.e. methylmethanesulfonate and ethylmethanesulfonate create many mutations

-G most prone to chemical damage

-human cell has 3.2x10^9 base pairs

depurination 10,000 times a day

deaminiation 100 times a day

1) incorrect base pairing (tautomers, wobble base pairing)

2) strand slippage during replication 

How is damaged DNA fixed? 

What are the DNA repair mechanisms? 

the cell has a DNA damage response that fixes damaged DNA, and if it can't fix the DNA, the cell undergoes apoptosis (cellular suicide).

MMR=mismatch repair

NER=nucleotide excision repair

BER=base excision repair

HR=homologous recombination pathways

NHEJ=nonhomologous end-joining pathway

-has to be strand specific, daughter strand must be repaired no the parent

- many cancers due to mutations in mismatch repair genes

multi-step process that corrects non-bulky damge to bases resulting from oxidation, methylation, deamination, or loss of the DNA base (apurination or apyrimidation) leading to abasic or AP sites. 

-glycosylases are class of enzymes that recognize specific types of damage. 

-most flexible and complex of the DNA repair pathways. 

-acts upon many types of DNA damage including pyrimidine dimers, bulky chemical adducts, DNA intrastrand crosslinks, some forms of oxidative damage

-process involves damage recognition, opening of DNA duplex around lesion, incision of the damaged DNA strand, gap repair synthesis, strnad ligation. 

-UV light causes photochemical reaction with thymine bases to form cyclobutane sturcture

-damage repaired by a light activated enzymes call Photolyases (not in mammals)

aka - crossover, occurring normally during meiosis providing greater energetic diversity

-recombination process uses the sister chromatid in the cell as a template

-portion of DNA is transferred from the template to the break on one strand and the ends ligated

-DNA polymerase, then uses repaired strand as template to repair other strands

-crossover produces new chromosomes that are half old and half new. 

Nonhomologous End Joining 

1) ionizing radiation or chemicals causes DSBs in DNA 

2) Ku70/Ku80 sense break and rapidly bind DNA ends

3) KU70/Ku80 slide along the DNA molecule and attract the catalytic DNA-PKcs (DNA dependant protein kinase) 

4)DNA-PKcs is phosphorylated as several serine

5) Artemis protein binds to DNA-PKcs complex

6)Phosphorylation destabilizes DNA-PKcs, which dissociates from DNA and now en-processing enzymes ligase IV, XRCC4, XLF, and Rad51 proteins bind

7)DNA Ligase IV joins ends

8)Damage is repaired with varying levels of fidelity

-works best with blunt ends-if overhangs are present, they're removed

-for badly damaged DNA-as find with chemical or radiation damage, get "dirty" breaks.

-require more processing and thus more sequence loss

-repair is nonhomoogous; no parent to compare to 

1. DNA replication is known as what with respect to templates?
2. Describe where DNA replication starts.
3. Descibe directionality AND continuity of DNA replication.
4. Replication requires what of the DNA helix?

1. Semiconservative.
2. Starts at the "origin"
3. Replication is bidirecional and semidiscontinuous
4. It requires unwinding

What are Hoogsteen base pairs? Where are they observed?

Provide (show) examples

They are non-Watcon Crick base pairs, found in RNA and complex DNA structures (triplex and quadruplex).

Hoogsteen pairing uses N7 of a purine and the NH2 group, rather than N1 and the NH2.

What is the most stabilizing force in DNA? Which interactions are strongest? 

Name one other effect which provides stabilization.

π (pi) stacking and G-C interactions are stronger than A-T. Provides ~30 kJ/mol of stabilization.

Hydrophobic effects also provide stabilization.

Heating DNA strands to cause them to separate. 

It lowers the viscosity.

Because regions of A-T pairing are weaker, so the denaturing expands this way. 

G-C pairing do not unwind until higher (more energetic temperatures).

What is the melting temperature, T_m?

What happens to absorbance as temperature increases?

Temperature at which half the DNA is unwound (denatured).

Absorbance increases over a narrow temperature range--once one part of the structure collapses, the remainder does too.

1. Electrostatic repulsions between strands.

2. Entropy--increased conformational freedom rather than an ordered structure.

3. Stacking forces, which can be strong enough to even force single-stranded DNA into a helix.

Two strands separate and each single strand is copied to generate a complementary strand.

The Meselson-Stahl experiment used heavy nitrogen to grow cells. These cells were moved to a medium containing light nitrogen. These cells were analyzed and found to have a parental heavy strand and one new light strand. The next generation had two hybrid DNAs and two light DNAs.

Leading strand is synthesized continously 5' --> 3'

Lagging starnd is composed of Okazaki fragments, put down in short sequences of 5' to 3'.

Used audioradiography to image radioactive DNA. Used a tritium label in essence, which showed that replication was bidirectional.

Around 1,000 bp/sec

Opens up double-stranded DNA for the formation of replication fork.

Tends to be positively charged in the center (binds to the negative phosphates!)

It has three active sites.

It has both 3'-5' and 5'-3' exonuclease activities. This serves as a proofreading function.

Function in DNA repair.

DNA polymerase I also acts downstream on the lagging strand to remove RNA primers and replace them with DNA.

DNA Polymerase III has processivity.  How and why?

What is DNA Pol. III used for?

It has a sliding clamp which attaches it to DNA. Processivity is how long the enzyme remains bound to the DNA. 

It is the enzyme primarily used for DNA synthesis.

1. Active site shape will only allow for "fit" of proper Watson-Crick base pair.

2. Minor grove recognition of purine N3 and/or pyrimidine O2 by amino acids in active site.

In E. Coli, it consists of DNA unwinding protreins, priming complex (primosome) and two equivalents of DNA Polymerase III.

It is responsible for the mechanism of replication.

S phase.

It is at the end of this point you have duplication of chromosomes.

Sometimes (in circular DNA) there can be more or less than 10 bp per turn.

Enzymes called topoisomerases (gyrases) can introduce or remove supercoils.

The complex of DNA, histones, and non-histone proteins within the nucleus of eukaryotic cell.

This is what chromosomes are made of.

Heterochromatin refers to highly condensed region of a chromsome which is generally inactive for transcription.

Euchromatin refers to uncondensed region of a chromosome that is active for transcription.

Chromosomes active in transcription tend to concentrate where?

Chromsomes inactive in transcription tend to concentrate where?

Active --> Middle of the nucleus.

Inactive --> Periphery of nucleus.

reverse of noraml transcription, occuring in some RNA viruses, in which a sequence of nucleotides is copied from an RNA template during the synthesis of a molecule of DNA. 

very error prone and many mutations can occur

What are the three forms of RNA? 

What are similarities and differences in function? 

mRNA (messenger RNA), rRNA (ribosomal), tRNA (transfer RNA) 

All 3 forms participate in protein synthesis

All made by DNA-dependant RNA polymerases, through transcription

Not all genes encode proteins. Some encode rRNAs, tRNAs

Transcription is tightly regulated. 

What are the differences between DNA and RNA?

(4 of them) 

1. deoxyribose vs. ribose (structural difference)

2. Ribose has chemical effect due to extra hydroxyl group. (DNA is not susceptible to alkaline hydrolysis. DNA reacts with acid to hydrolyze purine glycosidic bonds.) 

3. Different bases. RNA has Uracil instead of Thyamine

4. RNA has many possible secondary structures (single strand allows for different conformations. Non-WC base pairing)

"cloverleaf" secondary sturcutre. 4 helices, 3-4 loops with 73-94 nucleotides. 

Can form H-bonds between distal secondary structual elements

1) polymerase binds nonspecifically to DNA with lower affinity and migrates along it looking for promoter. 

2) Sigma subunit recognizes promoter. 

3) There are different factors that bind to different promoters (with different sequences)

4) RNA polymerase holoenzyme and promoter form "closed promoter complex" 

Polymerase unwinds forming "open promoter complex" (much tighter bind)  

1) binding of RNA polymerase holoenxyme at promoter sites

2) initiation of polymerization 

3) chain elongation 

4) chain termination 

- 3' OH is nucleophile that attacks the nucleoside triphosphate 

- hydrolysis of pyrophosphate provides energy for the reaction; performed by Phyrophosphatase (enzyme that cleases PPi)

- cleavage of PPi releases 19 kJ/mol 

- requires a template

What are the differences between RNA polymerization and DNA Polymerization? 

- RNA initiate with a NTP, no primer required. 

- RNA has no proofreading, error rate of 1 in 10,000

- RNA (20-50 nucleotides/sec) is slower than DNA (1000 nucleotides/sec)

ATP and GTP 

(most RNAs begin with a purine at 5'-end)

RNA polymerases I, II, and III transcribe _RNA, _RNA, and _RNA genes respcectively. 

rRNA, mRNA, tRNA

*Pol III transcribes a few other RNAs as well

assume 200 b.p. of DNA and a histone is 100 angstroms across. 

200 b.p. = ~680 angstroms. 

DNA is compacted to 1/7 of its length!

Lys and Ser

Posttranslational modifications of hsitones affect the accessibility of the DNA

Can experience histone acetyl transferase (HAT) or histone deactylase (HDAC) 

acetylation of the lysine removes positive charges, reducing the affinity between histones and DNA, ultimately making it easier for RNA polymerase to access promoter region. 

deacetylation does the opposite, and represses transcription. 

DNA methylation

1) adds a methyl group to which bases? 

2) is done using what compound? 

3) has what affect?

1) adenine or cytosine

2) S-adenoxy-:-methionine (SAM)

3) methylation "silences" DNA. associated with cancer

Alanine structure?

pKa's?

letter codes? 

[image] 

pKa OH - 2.6   NH - 9.7

Ala/A

Arginine structure?

pKa's? 

letter codes? 

[image]

pKa OH - 2  NH₂ - 9  +NH₂ - 12

Arg/R

Asparagine structure and pKa's?

letter codes? 

[image]

pKa OH - 2.76   NH₂ - 8.76

Asn/N

Aspartic Acid structure and pKa's? 

letter codes? 

[image]

pKa OH - 1.95   NH₂ - 9.7 

Asn/D

Glutamic Acid structure and pKa's?

letter codes? 

[image]

pKa OH - 2.16   NH₂ - 9.58

Glu/E

glutamine structure and pKa's? 

letter codes? 

[image]

OH - 2.16    NH₂ - 9.00

Gln/Q

glycine structure and pKa's?

letter codes? 

[image]

OH - 2.34   NH₂ - 9.58

Gly/G

histidine structure and pKa's? 

letter codes? 

[image]

OH - 1.70  NH₂ - 9.09   NH - 6.04

His/H

Isoleucine structure and pKa's? 

letter codes? 

[image]

OH - 2.26   NH₂ - 9.6

Ilu/I

leucine structure and pKa's? 

letter codes? 

[image]

OH - 2.32   NH₂ - 9.08

Leu/L

lysine sturcture and pKa's? 

letter codes? 

[image]

OH - 2.15    NH₂ - 9.16    +NH₃ - 10.67

Lys/K

methionine structure and pKa's? 

letter codes? 

[image]

OH - 2.36    NH₂ - 9.08

Met/M

phenyalanine structure and pKa's? 

letter codes? 

[image]

OH - 2.18    NH₂ - 9.09 

Phe/F

proline structure and pKa's? 

letter codes? 

[image]

OH - 1.95      NH - 10.47    

Pro/P

pyrrolisine structure? 

letter codes? 

[image]

22nd amino acid forget it 

Selenocysteine structure and pKa's? 

letter codes? 

[image]

OH - 1.9    NH₂ - 10

21st amino acid forget it

Serine structure and pKa's? 

letter codes? 

[image]

OH - 2.13    NH₂ - 9.15 

Ser/S

threonine structure and pKa's? 

letter codes? 

[image]

OH - 2.2      NH₂ - 8.96 

Thr/T

tryptophan structure and pKa's? 

letter codes? 

[image]

OH - 2.38     NH₂ - 9.34

Trp/W

tyrosine structure and pKa's? 

letter codes? 

[image]

OH - 2.24    NH₂ - 9.04   phenol - 10.10

Tyr/Y

cysteine structure and pKa's? 

letter codes? 

[image]

OH - 1.91    NH₂ - 10.28   SH - 8.14 

Cys/C

Valine structure and pKa's? 

letter codes? 

[image]

OH - 2.27   NH₂ - 9.6

Val/V

1. catalysis (enzymes)

2. transport (hemoglobin transports oxygen in blood; transport ion across cell membranes)

3. storage (myoglobin; oxygen storage in muscle)

4. coordinated motion (muscle, flagella)

5. mechanical support (collagen)

6. protection (immune system - antibodies)

7. regulation and communication (hormones, receptors, gene activation and repression)

8. generation and transmission of nerve impulses

9. toxins (bacterial, plant, etc.)

What properties are crucial to the structure and function of amino acids? 

- stereochemistry

- relative hydrophobicity or polarity 

- H-bonding properties 

- ionization properties

- other chemical properties 

- alpha carbon is chiral

- ALL naturally occuring in proteins are L-isomers

(levorotatory)

- ALL are S about the alpha carbon

what is the form of amino acids at pH 7? 

(think of pKa of carboxyl and amino groups)

pH = (pKa₁+pKa₂)/2

-using pKa's on either side of the neutral species 

- glycine (gly/g) 

- alanine (ala/a) 

- proline (pro/p)

- isoleucine (ilu/i) 

- valine (val/v)

- leucine (leu/l)

- methionine (met/m)

these AA are found in the interior of the membrane/proteins 

what amino acids are bulky and neutral? 

where are they found? 

- phenylalanine (phe/f)

- tyrosine (try/y)

-tryptophan (trp/w)

these AA are found in the interior of the membrane/protein

what amino acids are hydrophilic, neutral, H-bonding? 

where are they located? 

- serine (ser/s)

- threonine (thr/t)

- asparagine (asn/n)

- cysteine (cys/c)

- glutamine (gln/q)

these AA are found at the ends of the membrane/protein that are exposed to water

waht amino acids are acidic? 

- aspartic acid (asn/d)

- glutamic acid (glu/e)

- lysine (lys/k)

- arginine (arg/r)

- histidine (his/h)

Phe, Tyr, and Trp absorb UV light. Can allow for the determination of protein concentration. 

NMR spectra are characteristic of each residue in a protein and measurements can be used to elucidate 3D structures of proteins. spectra can get complicated fast...

- linear arrangement of n amino residues 

- polymers of 2, 3, a few, and many AA residues are called di-, tri-, oligo-, polypeptides 

- proteins consist of one or more polypeptide chains

- N-terminus end is the end of the chain with +NH3R group

- C-terminues end is the end of the chain with COO- group

the amide linkage formed between two amino acids resulting in the net release of a molecule of water. 

covalent chemical bond between carboxyl group of one molecule reacts with the amino group of the other molecule. 

- resonance of peptide bond induces a small electric dipole

- virtually all peptide bonds in proteins occur in trans configuration. except in proline

- resonance causes the the carboxyl, amino, and their respective alpha carbons to be in the same plane and rigid

- sequence differences arise from the various R groups attached to each alpha C (20 different AA to choose)

what can be said of the sequence of amino acids? 

- unique characteristic of every protein 

- encoded by the nucleotide sequence of DNA; form of genetic information

- read from n-terminus (left) to c-terminus (right)

- generally proteins contain > 40 residues (minimum needed to fold into tertiary structure)

- usually 100-1000 residues 

- percent of each AA varies 

Redox reactions forming a disulfide bond. 

[image]

in amino acid reactions what are the sites of modification? 

what are the reactions dependent upon? 

- sites of modification are the carboxl group, amino group, and side chains

- reactions depend on solvent pH, temp, ionic strength

nucleoprotein? 

hemoprotein? 

- are proteins that contain a nucleic acid derivative ofriboflavin: the flavin adenine dinucleotide (FAD) or flavin mononucleotide (FMN).

- Flavoproteins are involved in a wide array of biological processes, including, but by no means limited to, bioluminescence, removal ofradicals contributing to oxidative stress, photosynthesis, DNA repair, and apoptosis

what is synapsis? 

chemical reactions that revers the damage, returning the DNA to its proper state. 

include MMR, BER, NER, and more

a functioning unit of genomic DNA containing a cluster of genes under the control of a single regulatory signal or promoter

genes for enzymes for pathways that are grouped in clusters on the chromosome 

a regulatory sequence adjacent to an operon, determines whether or not the operon is transcribed 

segment of DNA which a transcription factor protein binds

what is positive gene regulation? 

turning on of the structural gene expression by the active repressor protein. the repressor protein alone can not bind to the operator and thus facilitates the binding of the RNA polymerase and transcription 

[image]

- in eukaryotes transcription is regulated by chromatin structure 

- in eukaryotes the positive mechanism is more important

- in eukaryotes transcription and translation occur at distinct times and places 

they can be spread out across 10-20kb of DNA upstream of the TATA box

some signals can be downstream of the coding gene, or even within introns

accomplish this by atomic contacts between protein residues and bases and sugar-phosphate backbone of DNA 

most contacts are in the major groove of DNA. 

zince finger (Zn-finger)

helix-turn-helix (HTH)

leucine zipper (bZIP)

[image]

- all bind as dimer to dyad-symmetric sites on DNA

- all contain 2 alpha helices separated by a loop with a beta turn 

- C-terminal helix fits in major groove of DNA; N-terminal helix stabilizes by dydrophobic interactions with C-terminal helix

- Helix 3 reads DNA sequence through H-bonds in major groove[image]

- two main classes, C₂H₂ and C_x

-  C₂H₂ domain consist of Cys-x₂-Cys and His-x₃-His. separated by at least 7-8 aas

-  C₂H₂ form a beta fold strand and an alpha helix that fits into DNA major groove

- C_x domain consist of 4, 5, or 6 Cys resifues separated by various numbers of other residues 

- leucine zipper proteins (bZIP proteins) dimerize, as homo- or hetero-dimers

- basicr region is DNA-recognition site

- homodimers recognize dyad-symmetric DNA

- heterodimers recognize non-symmetric DNA

DNA -- (transcription)-->  RNA --(translation)--> Proteins 

--(folding?)--> Functional Protein --(degradation)--> amino acid

prof. as Washington. 

invented Foldit and Rosetta

1930's-1940's was a theoretical biochemist and mathematician. 

showed hydrophobic residues and molecular forces caused the exclusion of water serve as nucleation points. (local energy minima must be avoided.) 

- hydrophobic amino acids interior

- hydrophilic amino acids of surface 

-satisfy H-bonding of backbone to exclude water

Levanthol's Paradox 

ex. 100 amino acids would take 4 x 10⁹ years to fold (if folding is random), therfore must be a pathway. 

because of the very large number of degrees of freedom in an unfoldedpolypeptide chain, the molecule has an astronomical number of possible conformations. Therefore "protein folding is sped up and guided by the rapid formation of local interactions which then determine the further folding of the peptide; this suggests local amino acid sequences which form stable interactions and serve as nucleation points in the folding process"

proposed by Ken Dill and Jose Onuchic

theory assumes that a protein's native state corresponds to its free energy minima under the solution conditions usually encountered in cells. 

although energy landscapes may be "rough", with many non-native local minima in which partially folded proteins can become trapped, the folding funnel hypothesis assumes that the native state is a deep free energy minimum with steep walls

by unfolding it. denaturation. 

can be done with heat, acid, base, detergent, or organic solvents

small proteins < 1 ms

others seconds to a minute

others require chaperones

1) hydrophobic collapse, water removal. ~1 ms

2) Formation of Secondary Structure 

alpha helix > beta sheet 

ns             ms

3) formation of weak tertiary contacts 

molten globule 

4) final tertiary contacts 

5) quarternary structures 

- interactions between monomers -> oligomers 

- non covalent 

- large surface areas interacting (+200 A) -> stability 

- identical or non-identical subunits (dimers, trimers, etc)

- proline isomerization 

- disulfide bonds 

- prosthetic group insertion 

- protein processing (post translational modifications)