Section 4: Nucleic Acids

What do nucleic acids consist of? What is it defined by?

They consist of bases linked to a sugar phosphate backbone

• form of linear information

• Each nucleic acid is defined by the sequence of bases

• Each monomeric unit contains a sugar, base and phosphate

What are info,fed in the formation of the nucleic acid backbone on the sugar? What’s the difference between DNA and RNA?

• 3’ OH and 5’OH

• DNA lacks 2’OH

How are sugars linked? What’s the directionality?

• they’re linked by phosphodiester linkage, 5’-3’

• The 2’OH in RNA can hydrolyze the phosphodiester backbone

What are purines vs pyrimidines?

Purine (double ring) - adenine, guanine

Pyrimidine (single ring) - cytosine, uracil, thymine

What’s a nucleotide?

Consists of a base, pentose sugar, one or more phosphates (nucleoside joined to phosphoryl group by ester linkage)

Ex. Adenylate = nucleotide

What’s a nucleoside?

Only the base and pentose sugar

Ex. Adenosine

How many bases per turn of double helix? How long? How much each base separated by?

34 Angstroms, 10.4 bases per turn, each base is 3.4 angstroms and 36 degrees

Four properties of double helix chain?

1. Two DNA chains of opposite directionality intertwine to form a right-handed double helix

2. Sugar-phosphate backbones are on outside, bases are inside

3. Bases perpendicular to axis of helix with bases separated by 3.4 angstroms and 10.4 bases/turn

4. Helix is 20 Angstroms wide

How many hydrogen bonds does AT and GC have?

GC-3

AT-2

What causes variations in base pairing?

• rotation of a base in RNA or DNA ends (reverse Watson crick)

• Tautomerization (wobble)

• Rotation around C1-N glycosidic bond (Hoogsteen)

What contributes to the stability of the helix?

1. Hydrophobic effect: hydrophobic interactions drive bases to inside of the helix, more polar residues outside

2. Base stacking: stacked bases attract each other through van der waals forced

• many weak non covalent interactions provide stability but still allow for strand separation

What is the major and minor grooves?

• proteins recognize patterns of H-bond donors/acceptors in the major and minor grooves

• Adenine-Thymine = (NHO/NO)

• Guanine-Cytosine = (NOH/NHO)

Why does the major and minor grooves exist?

Glycosidic bonds of each base in a pair are not diametrically opposite each other

Lined by hydrogen bonds and acceptors

What are supercoiled structures?

• Relaxed circle (260 bp, 10.4 bp/turn, 25 turns

• Negative superhelix (23 regular turns and two negative turns

• Unwound circle

  • Supercoiled species travel fastest through agarose gel

What does 3D RNA structure allow for?

Some RNA act as catalysts (ribosomes)

How does DNA replicate?

DNA polymerase: catalyses addition of deoxyribonucleotides to a DNA strand

E Coli polymerases=

• polymerase I: primer removal, DNA repair

  • 3-5 exonuclease

  • 5-3 exonuclease

• Polymerase III: replicative polymerase

  • 3-5 exonuclease domain

What induces a conformational change in DNA pol?

• binding of the correct incoming dNTP

  • Mg2+ stabilizes negative charges on phosphates, dNTP is added and helix clamps down on active site

What does DNA pol III catalyze during replication?

-strand elongation reaction

• Mg2+ stabilize negative charges and deprotonates hydroxyl so it can attack alpha phosphate

• Pyrophosphate is released, two bases are now connected by phosphate group

What would happen if ddNTP was added instead of dNTP?

It would still base pair and the O would bind phosphate of previous dNTP, however synthesis would stop following the ddNTP since there would be no 3’ hydroxyl to attach alpha phosphate

5 requirements/abilities of strand-elongation reaction by DNA pol

1. Requires dNTPs and Mg2+

2. New DNA strand is assembled on a pre-existing strand

3. Requires a primer to begin synthesis

4. Elongation is in 5-3’ direction

5. Can correct mistakes using 3-5’ exonuclease activity (proofreading)

How are mistakes corrected by Polymerase?

3-5 exonuclease domain removes incorrect nucleotides from 3’ end of the growing strand by hydrolysis

Mismatch results in a stall

Pause gives additional time for the incorrect region to flop into the exonuclease active site where it’s removed

What are the four components of DNA replication in E Coli?

• Origin of Replication (OriC): region of DNA that is the start site of replication

• DnaA: binds to OriC to initiate pre-priming complex

• SSB: single strand binding protein that keeps 2 single stranded regions separate

• DnaB: helicase that seperates duplex DNA

How do helicases unwind ddDNA?

Hydrolysis of ATP causes ratcheting of the subunits of the hexamer, pulling ssDNA through the center

What does primase do?

• rna pol that creates a short primer (10 nt) (directs dna pol where to go)

Describe the DNA replication fork

Parental DNA is getting unzipped by helicase, and held separate by SSB proteins

DNA pol works in 5’-3’ direction, so one strand can be synthesized continually (leading strand)

Whereas the other is synthesized in fragments (lagging strand), as helicase moves forward primase has to lay new primers for dna pol III

What does B2 do?

Sliding clamp improves processivity

Describe process of leading and lagging strand synthesis?

Leading strand:

• made continuously in 5-3’ direction by pol III

Lagging strand:

• looped out

• Starting from an RNA primer, pol III adds ~1000 nt in 5-3’ direction

• Released B2 sliding clamp

• New loop formed, sliding clamp added

• Primase creates new RNA primer

• Pol III synthesizes new Okazaki fragment

• Pol I fills gaps between fragments and removes RNA primer with 5-3’ exonuclease activity

• Ligase seals fragments

How does ligase join fragments

Joins the 3’ OH and the 5’ phosphate group of two fragments (with ATP)

Describe the process of ligase sealing fragments

Initial charging:

• catalytic lysine attacks the alpha phosphate of ATP, generating Lysyl-AMP adduct and releasing pyrophosphate

Transfer:

• adenylated ligase transfers AMP from lysine to the 5’ phosphate at the nicked backbone

Phosphodiester bond formation:

• 3’OH at the nick site attacks adenylated 5’ phosphate, making a phosphodiester bond and releasing AMP

What are mutagens? How can DNA be damaged?

• mutagens = chemical agents that alter DNA bases

• DNA bases can be a,termed through oxidation, sea inaction, alkylation, uv radiation, x-ray exposure

What is guanine oxidation?

• hydroxyl radical reacts with guanine to form 8-Oxoguanine

• 8-oxoguanine base pairs with adenine; mismatch since one strand will have AT base pair

What’s cytosine deamination?

• occurs around 500x per cell/day

• Removal of an amino group through the reaction with water

• Cytosine + water → uracil + ammonia

What’s adenine deamination?

• removal of amino group with water → hypoxanthine

• Hypoxanthine base pairs to cytosine; AT→ CG

What’s 5-methylcytosine Deamination

• cytosine is methylated at C5 to regulate gene transcription

• Deamination of 5-methylcytosine results in thymine

Can thymine undergo deamination?

No, thymine lacks an exocyclic amine group

What’s guanine alkylation?

• alkylation = addition of hydrocarbon to the base

• Alfotoxin B1 (crop mold) epoxide alkylates N-7 of guanine

• Epoxide = very reactive 3 atom ring structure

• GC→AT transversion

What’s a thymine dimer?

• UV lights covalentky links adjacent pyrimidines along the dna strand

  • pyrimidine dimer (cyclobutane ring) that creates a bulge in the DNA double helix

What are 3 principles of DNA repair?

1. Damage and repair is occurring constantly

2. Often repair will restore genetic info

3. Sometimes, it is not possible to restore original info so the cell uses approximate repairs and or undergoes apoptosis

What are the three steps for DNA repair

1. Recognize the damage

2. Remove the damage

3. Repair the damage

What is mismatch repair?

• Mismatch is recognized by MutS

• MutL binds and recruits MutH (endonuclease)

• An exonuclease excises incorrect region

• DNA pol III fills the gap

• DNA ligase seals the backbone

• Eukaryotes and prokaryotes

What’s direct repair?

• repairs without removing fragments of DNA

• Photochemical cleavage of pyrimidine dimmers by DNA photolyase

  • uses energy of visible light to break cyclobutane ring! Doesn’t require nucleotides

  • Not in mammals

What’s nucleotide excision repair?

• “dark repair”, doesn’t require blue light

• E. coli, eukaryotes, mammals

• Recognizes distortions in helix like bulge created by pyrimidine dimer

• UvrABC excinuclease cuts DNA at two sites

• DNA pol I fills the gap

• DNA ligase repairs the phosphodiester backbone

What’s base excision repair?

• non helix distorting damage

1. Defective base flipped into DNA glycosylase active site (goycosidic bond cleavage)

2. AP endonuclease nicks phosphodiester backbone

3. Deoxyribose phosphodiesterase removes deoxyribose phosphate unit

4. DNA pol I inserts correct nucleotide

5. DNA ligase seals strand (creation of phosphodiester backbone)

What is needed for transcription to happen?

• Template DNA

• NTPs (ATP, GTP, UTP, CTP)

• RNA polymerase

• Mg2+

What are the subunits and function of E. Coli RNA polymerase

• alpha 1 and 2: Assembly of core enzyme; interacts with regulatory factors

• Beta and beta’: Catalysis, interactions with DNA, RNA

• w: required for structure/folding

• sigma 70: promoter recognition

How is transcription in E. Coli initiated?

• Sigma 70 decreases affinity of RNA pol for DNA

• Sigma 70 recognizes and binds the promoter sequence (-35 consensus sequence and -10 pribnow box TATAAT)

• After several nucleotides of RNA are synthesized, sigma falls off the core enzyme

• NO primer needed

When does elongation begin?

• after formation of the first phosphodiester linkage

What’s the elongation reaction for RNA transcriptase?

• 3’ ON of terminal ribose of growing chain attacks inner most phosphoryl group of the incoming ribonucleotide triphosphate

• Synthesis begins de novo; no primer needed

How is transcription intrinsically terminated?

• Hairpin in RNA product causes RNA pol to pause

• rU-dA base pairs are weak, so that RNA dissociated from DNA template and enzyme

• Protein dependent termination = Rho Protein (helicase)

What are differences in transcription between eukaryotes and bacterium?

• translation and transcription occur simultaneously in the cytoplasm

• Simple control elements

• Termination signal is GC rich hairpin poly U structure (not polyA tail)

• mRNA is not transported across a membrane (nuclear envelope in eukaryotes)

What are promoter elements for eukaryotic RNA pol II

• cis acting element = DNA sequences that regulate expression of a gene located on the same molecule of DNA

• TATA -25

• Inr = initiator +1

• DPE = downstream core promoter element +30

• Enhancer (can be more than 1 kb from start site)

• CAAT box and GC box (-40 to -150)

What’s the Pre-Initiation Complex?

• Transcripotion factors bind cis-acting elements to recruit RNA polymerase II

• TATA box is recognized by TFIID by the TATA binding protein

• TFIID is a dynamic protein complex

What are the 5 steps of forming the pre initiation complex

1. TFIID binds DNA and the TBP domain moves between multiple conformation until it binds the TATA box

2. TFIIA binds and stabilizes the complex and promoter region

3. TBP fully engages promoter, bending DNA

4. TFIIB recognized the TBP/DNA complex and recruits Pol II and TFIIF and TFIIE

5. TFIIH (helicase) unwinds DNA and phsophorylates the C-terminal domain of pol II, which triggers the elongation and recruitment of RNA processing enzymes

Does Pre-Initiation Complex initiate transcription at high levels? How much influence does one TF have?

• PIC initiates transcription at low levels

• TFs that bind other sites stimulate high levels of transcription

• One TF has little influence, many are needed to form a complex that influences transcription (combinatorial control)

How is the mRNA transcript modified?

• a 5’ 7-methyl guanosine cap

• A 3’ polyadenylate tail

What occurs during splicing?

Introns (non coding regions) are removed and exons (coding regions) are joined together

When does the 5’ cap occur?

Very early on in transcription, after 20-30 nucleotides of RNA are synthesized

what is the cap synthesizing complex composed of?

Four enzymes that associate with phosphorylated RNA pol II

What purpose does the 5’ cap serve?

• protect from degradation by nucleases

• Interacts with ribosome to enhance translation

What’s the steps in adding the 5’ cap?

1. Removal of the terminal gamma phosphate at the 5’ end by phosphohydrolase

2. Diphosphate 5’ end attacks alpha phosphate of a GTP to form a 5’ to 5’ triphosphate linkage

3. NT of guanine is methylated

4. Methylation of 2’OH of adjacent riboses

What is the poly A tail?

• ~ 250 adenylates at the 3’ end of the RNA

What are the enzymes involved in building the poly A tail associated with?

C terminal domain of RNA pol II

What role does PolyA tail have?

Enhance stability and translation efficiency (and nuclear transport)

How is the polyA tail added?

• CPSF (cleavage and polyadenylation specificity factor) recognized the cleavage signal and an endonuclease cleaves the mRNA transcript

• Poly(A) polymerase adds ~ 259 adenylate to the 3’ end

How is the splice site marked?

Introns begin with GU and ends with AG, conserved from yeast to humans

What’s the spliceosome?

• complex of protein and RNA that excises introns and joins exons

What type of reaction does spliceosome catalyze?

• 2 transesterification reactions (reaction of an alcohol and ester to make a different alcohol and ester)

Describe the splicing process

• branch site adenylate 2’OH attacks phosphoryl at 5’ splice site

  • exon 1 is released

  • Lariat intermediate is formed

• 3’OH of exon 1 attacks phosphoryl group at the 3’ splice site

  • splices product exon1-exon2

  • Lariat intron

What is the spliceosome made up of?

• ~300 proteins and five critical small nuclear RNAs (snRNA)

  • U1,U2,U4,U5,U6

  • SnRNA + protein = snRNPs (small ribonucleoprotein)

Where does U1 and U2 bind

U1 binds the 5’ splice site

U2 binds the branch site

What does U4,5,6 do?

• the U4-U5-U6 complex joins and displaces U1 and U4

  • Extensive interaction between U2 and U6 brings the 5’ splice site and branch site close together

What facilitates the first and second tranesterification?

• 1st = facilitated by the “catalytic core” of the spliceosome

• 2nd = facilitated by U5; requires Mg2+ for catalysis

What products are formed from this?

• spliced RNA product and lariat intron