General Exam Review Topic: DNA

v. Nucleic acid (such as deoxyribonucleic acid) is chain of NTs.

vi. DNA written 5' à 3'

vii. One 360° helical turn = 10 base pairs (“Repeat”) = 3.4 nm or 34Å (“Pitch”)

viii. Distance from one base to adjacent base in the helix = 3.4Å (“Rise”)

ix. Bases are not exactly on central axis of the helix, this results in the major & minor grooves

x. Hydration requirement

1. Hydration very important for conformation and utility of nucleic acids

2. Strength of these aqueous interactions is greater than for proteins due to their highly ionic character

3. Hydration is greater and more strongly held around the phosphate groups that run along the inner edges of the major grooves.

4. Hydrating water is held in a cooperative manner along the double helix in both the major and minor grooves, which aids both the zipping (annealing) and unzipping (unwinding) of the double helix.

5. Altering hydration can change DNA structure.

e. Gene is segment of DNA corresponding to a single protein (or single catalytic or structural RNA molecule)

i. 30,000 genes in human genome – allows efficient TXN. Expression of individual genes is regulated. Stretches of regulatory DNA are interspersed among coding segments. Noncoding regions bind to proteins that ctrl TXN.

ii. 477 genes in smallest known genome – Mycoplasma genitalium

iii. 200-300 genes minimum # for a viable cell in the environment today.

iv. Cute analogy – DNA is made up of letters (NTs), these letters make words (seq for mRNA codons), these words make sentences (genes)

v. Genes

1. Function: DNA à mRNA à protein à mature/ modified protein

2. Structure: Upstream regulation sites à promoter (TATA) TXN initiation site à 5' UTRà Exon à Intron à Exon à 3'UTR à Poly A add site.

3. Splicing: 3 vital positions on RNA. 5' splice site, 3' splice site & branch point in the intron seq that forms the base of the excised lariot. Endonuleolytic cleavage of introns and fusion of adjacent exons (performed by snRNAs+ 7 protein subunits = snRNPs = core of the splicosome that performs pre-mRNA splicing).

4. Note: 3' end of intron = splice acceptor, 5' end of intron = splice donor

f. Telomeres

i. 5-15 kb repeat of TTAGGG bound to proteins

ii. Shortens 100 bp / division

iii. Length maintained by telomerase: sees G at tip, elongates 5'à3'. It synthesizes a new copy of the repeat using an endogenous RNA template+primer. Telomerase reverse transcribes DNA. DNA POL completes synthesis of remaining lagging strand.

g. Repetitive DNA

i. Interspersed repeats (SINES-10% genome, LINES-10% genome)

ii. Tandem repeats (VNTR, noncoding, often in heterochromatin)

III. Organization

a. Nuclear genome >30,000 genes, mitochondrial genome 37 genes

b. 35% genes, 65% extragenic

c. Of the genes: 5% protein encoding (1-2% of entire genome), 95% non-coding

d. Of extragenic: 40% mod to high copy repetitive, 60% low number repeat

e. Chromosomes

i. 22 pairs homologous chromosomes & 2 sex chromosomes

ii. Chromosome has centromere, telomere, & ori (classified by centromere location: acrocentric (near ends), submetacentric (midpt to end), metacentric (midpt)). Kinetochore is present at centromere for spindle attachment

iii. 6 ft DNA compacted 10,000 fold in the chromosome

iv. Packaging: Nucleosome (8 histones & 147 bp DNA form core)àfurther to chromatin fiber (30nm)àfurther to chromatid fibers.

v. Nucleosome organization: 2-H2A+2-H2B (outside), 2-H3+2-H4 (inside), 147 bp DNA, 1.7 left handed turns, central 80 bp in contact with H3 & H4; H1 binds outside – completes 2 helical turns = chromatosome.

vi. To digest it: (Remove nuclei of cell & expose to partially denaturing conditions). Chromatin = beads on a string. (Nuclease digest linker DNA) Nucleosome core particles. (Disassociation w/ high [salt]) octameric histone core + piece of DNA. Note: histone core has 142 H-bonds, hydrophobic interactions and salt linkages. Sequence specific DNA binding proteins

f. Chromatin compaction

i. Active state: acetylated DNA in nucleosomes. TFN factors and RNA POL can access the DNA.

ii. Compaction: DNA methyl-transferase methylates DNA. Histone deaceylase associates + methyl-CPF binding proteins & associated co-repressors. No aceylation, heavy methylation & tight winding = Transcriptional silencing.

IV. Types/ Conformations of DNA

a. A-DNA

i. Pitch = 28.2 Å, 11 bp

ii. Less common

iii. Right handed

iv. Base pairs closer to outer edge of helix

v. More compact than B-DNA, # residues per turn (11, more fit in one rotation than B-DNA)

vi. Narrow and deep major groove and very wide but shallow minor groove

b. B-DNA

i. Pitch = 34 Å, 10 bp

ii. predominant natural DNA

iii. Right handed

iv. Base pairs closer to the central axis

v. Wide and deep major groove and a narrow and deep minor groove and requires the greatest hydration. Partial dehydration converts it to A-DNA decreasing the free energy required for A-DNA deformation and twisting, which is usefully employed by encouraging supercoiling but eventually leads to denaturation. Further dehydration will result in the least hydrated D-DNA (favored by excess counter-ions ions that shield the DNA phosphate charges), which has a very narrow minor groove with a string of alternating water and counter-ions distributed along its edge

c. C-DNA

i. Pitch = 31 Å, 9.3 bp

d. D-DNA

i. Pitch = 24.2 Å, 8 bp

e. Z-DNA

i. Pitch = 43 Å, 12 bp

ii. Left handed, zig-zag path

iii. Has even more residues per turn, most compact.

iv. Rise is greatest bc its rigid and stretched out

f. other conformations

i. stem & loops

ii. Octahedral structures

iii. Superhelicies/ coiled coil

V. Function

a. Nucleotides

i. Energy (ATP, GTP – donates P in rxns)

ii. Cofactors (NADH – donates an H in rxns)

iii. Structural (DNA & RNA – radox partners)

iv. Transfer RXNs (as ATP, charging AA to tRNA)

b. DNA

i. Replication

ii. Transcription (Template strand 3' à 5', Coding strand 5' à 3' , mRNA) 5'à 3'c

iii. DNA(neg) –protein (pos) binding

VI. Prokaryotic vs. Eukaryotic Genomes

PRO EUK

Circular Linear

No nucleosomes (has nucleoid loops) nucleosomes à chromatid fiber

No introns Has introns

Gene dense 65% extragenic

No repeats Tandem repeats & interspersed repeats

No telomere Telomere & telomerase

No splicing Splicing

Gene clustering (mult Ps on 1 gene) 1 protein product on one gene

~1800 protein coding genes 30,000 genes

1 replication site multiple replication sites

2-4 Mb genome >3,000 Mb genome

VII. Applications in Science

a. Cleavage of DNA at specific sites by restriction nucleases, which greatly facilitate the isolation and manipulation of individual genes

i. Cleavage sites are usually palindromic & 4-8 NTs long. They can cleave blunt ends or cohesive/ staggered ends.. 1^st few letters tell the organism the RE is from (ie. EcoRI is from E.coli). Bacterial genome is protected from RE activity by methylation at As & Cs.

ii. DNA produced by splicing together two or more DNA frags = recombinant DNA.

iii. Can also generate a collection of plasmids called a cDNA/ genomic DNA library by shotgun RE treatment to produce many gene frags of the genome or cDNA made by RT from mRNA. The rags are ss à ds DNA by DNA polymerase and are inserted in plasmid or virus vector and cloned. cDNA libarary is specific to type of cell used to prepare the library.

b. DNA cloning either through the use of cloning vectors or PCR, where a single DNA molecule can be copied to generate many billions of identical molecules

c. Nuclei acid hybridization, which makes it possible to find a specific sequence of DNA with good accuracy and sensitivity on the basis of its ability to bind a complementary NA sequence.

i. Isolated DNA can be labeled with radioisotope ³²P to make traceable probes.

d. Rapid sequencing of all NTs in a purified DNA fragment, which makes it possible to ID genes and deduce the AA seq. of the proteins they encode.

Additional misc:

-          Oligonucleotide = A compound comprising a nucleotide linked to phosphoric acid. When polymerized, it gives rise to a nucleic acid.  A short nucleotide polymer.

-          Nucleosie analog = A synthetic molecule that resembles a naturally occuring nucleoside, but that lacks a bond site needed to link it to an adjacent nucleotide.

-          Note that nucleotides have only one phosphate group, whereas ATP has three, so ATP is not strictly speaking a nucleotide. AMP or adenosine mono-phosphate, is a nucleotide. AMP is also called adenosine 5'-phosphoric acid or adenylic acid.  AMP contains a ribose group as its sugar, while dAMP, or deoxy-AMP, contains a deoxyribose as its sugar.

-          NTs present in RNA are AMP, GMP (guanylic acid, or guanosine 5'-phosphoric acid), CMP (cytidylic acid, or cytidine 5'-phosphoric acid) and UMP (uridylic acid, or uridine 5'-phosphoric acid).

-          NTs present in DNA are dAMP, dGMP, dCMP and dTMP (deoxythymidylic acid, or deoxythymidine 5'-phosphoric acid)

-          4 nucleosides of DNA are deoxyadenosine (dA), deoxyguanosine (dG), deoxycytosine (dC), & (deoxy) thymidine (dT, or T).

-          Nucleotides are acidic, hence the name.

-          The carbon atoms of the deoxyribose sugar are numbered 1', 2', 3', 4', and 5' to distinguish from the numbering of the atoms of the purine and pyrmidine rings.

-          DMT (dimethoxytrityl protecting group) can be added, instead of a phosphate group, to block DNA synthesis & 3’ end tie up Carbon with triple bond to a nitrogen

-          The deoxyribose sugars are joined at both the 3'-hydroxyl and 5'-hydroxyl groups to phosphate groups in ester links, also known as "phosphodiester" bonds.

-          Proteins that bind DNA (Topoisomerases, Histones, RNA POLY, Negative regulation – repressors (ie. lac repressor), Positive regulation – activators (ie. CFP), Attenuating proteins

NT = nucleotide

TXN = transcription

AA = amino acid

RXN = reaction