BMS 321 Exam 3

What are the Structural Characteristics of Nucleotides

-5 Carbon sugar

-Nitrogenous base attached to 1’ carbon by glycosidic bond

-Phosphate group attached to a 5’ carbon

How to Form a Nucleic Acid

Nucleotides linked by phosphodiester bonds.

5’ phosphate linked to 3’-OH.

This creates a negative sugar phosphate backbone. Repels Nucleophiles

Structure and Important Characteristics of DNA double helix

Right hand double helix. 2 anti parallel strands. Bases inside and backbone outside. 36 degrees rotation per base. Bases are perpendicular to axis. Bases form hydrogen bonds across helix. Helix is 20 A wide.

Base Stacking

Stabilizes helix using hydrophobic effect to drive the bases inward. Also stabillizes through van der waal forces.

Separating Strands

Denaturing. Has to happen during replication or transcription.

Major and Minor Grooves

Form because glycosidic bonds are not directly opposite. Allows proteins to access HB donors and acceptors on base edges.

A and B form

B form is watson Crick model and most common. A form is shorter and wider, bases are tilted, and has RNA double Helices-RNADNA hybrids.

Z DNA

Left handed helix. zigzagged backbone. 10 z-dna proteins. Long and narrow. Often near GC rich sequences.

What is the structure and important characteristics of RNA

Single strand regions. Folds back and base pairs to make different structures. Non-Watson bp. Has stem loop.

Stem Loop

Double helix over short region. Unpaired bases in loop.

Complex RNA Structure

Widely separated bases. Unpaired from stem. Non-Watson often. Stabilized by Mg Ions.

Amount of Hydrogen bonds during base pairing

A-T is 2 bonds. G-C is 3 hydrogen bonds.

How are DNA and RNA different

DNA: Deoxyribose, ATGC, double strand, more stable, genetic info, AT and GC

RNA: Ribose, OH, AUGC, single strand, less stable, gene expression and regulation and catalysis.

E. Coli

5 million bases. 1000x longer than bacteria, circular chromosome, compacts the DNA. Twist into superhelix via supercoiling.

Packing Methods in Eukaryotes

4 meters of DNA in 46 linear chromosomes. Wind into higher order structures. Has histones, nucleosomes, histones, and chromatin. Compacts DNA by a factor of 7.

Histones

Proteins that bind and package DNA. Protein of chromatin. Highly conserved in eukaryotes.

Nucleosomes

DNA and histones combined. has linker DNA which will join nucleosomes. Histone H1 binds to linker DNA.

Histone Octomer

2 copies of each H2A, H2B, H3 and H4

Histone H1

Binds near the DNA entry and exit sites of the nucleosome and helps stabilize chromatin structure.

How do Histones and DNA Interact?

N-terminal tail extends from nucleosome core structure. Histone protein has a tail that wraps around histones. These tails are rich in Arg and Lys residues. Can change how tight DNA is packaged. Also Electrostatic interaction: Backbone is negative and tails are positive which stabilizes DNA packaging.

Cis-Acting Sequence

Regions of DNA or RNA that help regulate transcription, replication, and RNA splicing on that same nucleic acid. Actual sequence will vary from site to site. Determines optimal sequence by comparing multiple. Consensus sequence: most common residue at each position.

Trans-acting Factors

Protein that regulated process on a different nucleic acid by binding. Bind to consensus sequences. Actual sequence can vary from the consensus but the closer it is the better binding and the stronger the effect.

Rules for making Nucleic Acid Strands

Watson Crick Basepairing, Using a template strand, template and new strand are antiparallel, and new bases are added to the 3’ end.

Chemistry of how we add a nucleotide

3-OH group attacks 5’-PO4 group of incoming nucleotide. Need free 3’-OH group which requires a primer.

Releases Pyrophosphate which is hydrolyzed to Pi.

DNA Replication

Occurs before division, need exact copy, and needs to happen quickly. Sequence of one strand determines the sequence of partner. Separate two strands and synthesize complementary seq. to generate 2 identical daughter helices (One parent strand and one new one. Semiconservative replication).

Meselson Stahl Experiment

Grow bacteria in media with heavy N isotope 15N. Change to growth media with 14N. Examine DNA with density gradient eq. centrifugation. (Gensity gradients to cesium chloride which forms bands that can be detected using UV light absorbtion). Generation 0 was all heavy. Generation 1 was all intermediate. Generation 2 was half intermediate and half light.

Dispersive Model

Parental DNA dispersed throughtout the daughter molecule randomly.

DNA Polymerase

Needed for DNA Replication

I - Primer removal and DNA repair.

II - Repair and attachment of bulky hydrocarbons to bases

III - Replication.

IV and V - Repair

Thumb Domain

Holds DNA in place and keeps DNA polymerase attached to the dna as it moves along

Fingers Domain

Helps position the incoming nucleotide so it can pair correctly with the template strand.

Palm Domain

Contains the active site where the chemical reaction occurs that adds nucleotides to the growing dna strand.

What is needed for DNA synthesis?

DNAP, 4 dNTPs, MG (to neutralize negative charge), template strand, and nucleotides added to 3’ end.

Initiation of replication

Rapid and coordinated among more than 20 proteins. occurs at the origin.

OriC Locus

Origin of replicaiton in E. Coli. unique 245 bp site. binding site for DnaA. 3AT rich regions.

DnaA and B

A - binds to oriC locus. recruits DnaB to unwind the DNA.

B - Binds at 3AT-rich sequences. Is a helicase.

Helicase

Enzymes that separate DNA strands. acts as a wedge to pry the helix apart. ATP hydrolysis causes a conformational change.

SSB

Single stranded binding protein prevents reannealing

Adding Primer

Short nucleic acid that provides free 3’-OH group. Needs primase

Primase

RNA polymerase. Makes short RNA primer complementary to DNA strand. Part of primasome and joins the prepriming complex.

Initiation

Forms prepriming complex, seperate DNA strands, add primer.

Elongation-Synthesize DNA

DNA polymerase adds nucleotides to growing DNA strands in the 5-3 direction. Both strands of parent DNA are templates.

Replication Fork

Y-Shaped region where the parental DNA strands seperate. moves along DNA in 1 direction as the double helix is unwound.

Leading and Lagging Strand

Leading- synthesized continuously in the same direction that the replication for moves

Lagging: Synthesized discontinuously in short pieces called Okazaki fragments (each with their own primer) which are later joined together.

DNA Polymerase III

Enzyme for majority of DNA synthesis in E. Coli.

Multisubunit protein: 2 polymerase subunits. sliding clamp-B2

Rarely releases DNA template one it begins. Complex moves in 1 direction.

Leading strand synthesis

Primase-1 RNA primer

Helicase- unwinds DNA at replication fork.

DNA synthesized continuously by one polymerase subunit.

Lagging strand synthesis - Elongation

Primase- Primer fo each fragment

Loop lagging strand to pass through the polymerase active site in the 5-3 direction.

allows DNAP III complex to move in one direction

after 1000 nucleotides synthesized then the loop is released and a new one is formed
Primase will lay a new primer and SSB is used.

Lagging strand synthesis - Termination

DNAP I exonuclease activity removes the primers and polymerase activity to replace RNA with DNA. DNA ligase joins the fragments.

Topoisomerases

Enzymes that induce or eliminate supercoils.

DNA gyrase

Relax supercoils from unwinding of DNA

Specificity of DNA replication

Due to H bonding between the new nucleotides and the template. Also the overall shape of base. Enzyme changes structure when right dNTP binds: Induced fit. Finger domain closes over the nucleotide and forms pocket that only properly shaped nucleotides will fit.

Proofreading Mechanism

Removes incorrect base to increase accuracy. 3-5 exonuclease activity. incorrect base is not tightly bound which causes polymerase to pause and new DNA moves to exonuclease active site and mismatched base is cleaved off.

Cost of Editing

1/20 bases removed is correct.

Endonuclease

Cleave phosphodiester bond within a polynucleotide chain

Exonuclease

Cleave phosphodiester bond from end of polynucleotide

Excinuclease

Excise a section of DNA

Eukaryotic DNA replication

More DNA than bacteria. Linear molecule (chromosome) and multiple chromosome. Multiple origins of replication - each is a replication unit or replicon. humans have 30,000 replicons.

Licensing factors

Protein that allow one replication per replicon per round of DNA synthesis. In eukaryotes

DNA polymerase Alpha

Has primase activity, initiates DNA replication, generates 20 base DNA molecule

DNA polymerase Delta

More processive and extends DNA molecule.

Problems wit ends of linear DNA molecules

1. susceptible to damage by nucleases

2. Lagging strand will shorten at the end due to primer.

Telomeres

Solves end replication problem. 6ntd repeats at ends of chromosomes. G rich. One strand is longer than the other to form loop structure. protects the end.

How Telomeres solve end replication problem

Lose telomere repeat at the end of round of replication instead of important sequence. Eventually it would all be gone so telomerase is used to extend repeat.

Telomerase

Enzyme that extends telomere repeat. is a reverse transcriptase. RNA serves as a template for telomere repeat.

Telomerase Mechanism

Pair RNA template with DNA. Use reverse transcriptase to extend DNA strand. Lay down primer and DNa Polymerase fills in other strand.

Low level of Telomerase in Cells

Telomeres shorten over time. Signals cell death. Limits cell proliferation

High Level of Telomerase in Cells

Allow for unlimited proliferation, cancer cells. Target for chemotherapy.

What is PCR

Make copies of target DNA by directing DNAP to begin copying. Can study specific sequences when starting with very little. Has a lot of specifity and amplification. Just need flanking seqeuence. amplify targets of 10kb.

What is PCR used for

Research- detect mutations and produce DNA for Experiments.

Diagnostic - detect DNA or RNA from pathogens (need to reverse transcribe for RNA).

Forensics - Analyze DNA evidence to help identify or exclude suspects

What is Needed for PCR

Target DNA, primers, dNTPs, Mg, and Heat Stable DNAP.

Taq polymerase

DNA polymerase from Thermus aquaticus.

PCR RXN

repeat 3 steps till desired amplification.

1. dnaturation or melting- 94 degrees.

2. Annealing- cool to allow the primers to anneal to the DNA 52-58 degrees.

3. Elongation- heat to 72 degrees and allow the polymerase to synthesize DNA

Sanger Sequencing

Terminate replication with dideoxy nucleotides. set up 4 polymerase rxns. ddNTP is incorporated randomly and will halt replication. run on a gel and observe different fragments. fragment length corresponds to position of the base complementary to ddNTP. Fluorescent detection- each ddNTP has fluorescent label.

Gene

A segment of DNA that codes for RNA

Main Types of RNA

Not all RNAs encode proteins

mRNA(make proteins), tRNA, rRNA

RNAP

Enzyme that catalyzes transcription. requires ribonucleotide triphosphates and Mg or Mn. Quatnary structure.

Holoenzyme

RNAP that contains sigma subunit. initiates RNA synthesis

Core Enzyme

Does not have the sigma subunit. Elongates the RNA

Sigma Subunit

RNAP subunit. takes part in promoter recognition and binds to -10 and -35 elements. Allows holoenzyme to have decreased affinity for DNA to scan for promoter. Will dissociate from RNAP to find another core enzyme.

Strand for RNA synthesis

Template strand- dna read to make RNA

Coding strand- DNA strand with same sequence as RNA (T instead of U)

Chemistry of RNA synthesis

3’OH attachs 5’phosphate group. DOES NOT REQUIRE A PRIMER

Initiation of transcription

Promoter is 1000 bases around the start site. it positions RNAP at initiation site. Two consensus DNA sequences makeup promoter region (-10 sequence AT rich and -35 sequence). next step would be to go from open to closed promoter complex.

Promotor Strength

Stronger promotor- better able to bind sigma subunit. closer to consensus seq. and optimal spacing allow this.

other seq newar the promoter can alter effectiveness.

Transcription Factors

Regulatory proteins that bind to consensus seq to increase or decrease promoter activity.

Closed Promoter coplex

DNA helix is not unwound

Upstream Element

Transcription factor. bound by alpha subunit of RNA polymerase. Increases efficiency-adds binding site for RNAP. found in highly expressed genes.

Open promoter complex

RNA polymerase unwinds 17 bases of DNA for template.

Elongation

Sigma factor dissociates after 9-10 bases. core enzyme binds tightly to DNA, rate is about 50 bases per second.

Transcription Bubble

Region with RNA polymerase, unwound DNA, and RNA product. DNA-RNA hybrid of 8 nucleotides. RNA exits from the complex.

Termination

Signal in newly transcribed RNA-2types

intrinsic or protein dependent.

Both stop nucleotide incorporation, RNA-DNA hybrid dissociates, DNA reanneals, and RNAP releases DNA

Intrinsic Termination

Forms a stable stem loop followed by U residues.

Stem loop-causes polymerase to pause

U Residues- less stable RNA-DNA hybrid

Allows RNA to dissociate from template to enzyme

Protein dependent Termination

Rho protein binds to RNA sequence. using ATP it will move towards DNA and Rho will run into RNAP and bumps it off the DNA and it terminates transcription.

Transcription in prokaryotes

Holoenzyme scans and binds promoter, sigma factor dissociates after 9-10 bases, core enzyme binds tightly to DNA to transcribe gene, then termination.

RNA Modification in Prokaryotes

mRNA is not modified

rRNA and tRNA are modified - structural and functional diversity

cleave from a large precursor, addition of nucleotides post transcription, and modify bases and ribose.

Antibiotics inhibit Transcription

Rifampicin-blocks exit of new RNA

Actinomycin D- intercalates between the bases of DNA

Default transcriptional state

ON

Operons

cluster of genes under control of a single promoter. Involved in the same molecular process.

Metabolism of Lactose

glucose is preferred food source. can use lactose when glucose is low. Beta-galactosidase catalyzes metabolism of lactose(minimally transcribed unless lactose is present)

Lac Operon

3 genes needed for lactose metabolism. Lac Z, Y, and A. all 3 are expressed from one mRNA (polycistronic transcript.)

Lac Z

b-galactosidase, breaks down lactose.

Lac Y

Permease, transports lactose across the cell wall.