DNADeoxyribonucleic Acid

What is DNA?• DeoxyriboNucleic Acid (DNA)

• genetic information for all forms of life

• found in all living cells

• responsible for the production of inherited traits

• encodes, stores, & transmits instructions for making proteins

The Structure of DNALarge and complex, easy to understand due

to orderly, recurring structure.

The Basic Structure• DNA is a polymer .• made of just 4 simple compounds

– nucleotides.• Each nucleotide has a phosphate

group, deoxyribose (a sugar), and a nitrogenous base.

S=sugar, P=phosphate group,A=Adenine, G=Guanine, C=Cytosine, T=Thymine

Nucleotide BasesThere are only four different kinds of bases, which make up the complex genetic code.

• Adenine, Guanine, Cytosine, & Thymine. •Complementary base-pairing rules: 1. Adenine always pairs with Thymine. 2. Cytosine always pairs with Guanine.• Adenine & Guanine are purines (two C/N rings). •Cytosine & Thymine are pyrimidines, (one C/N ring). • approx. 3 billion base pairs in each somatic cell.

- a spiral shape used to describe the form of DNA.

•Resembles a twisted ladder wound around a cylinder - two nucleotide chains connected by hydrogen bonds between the nucleotides.

•Sugar-phosphate backbone - deoxyribose sugars covalently bonded to an adjacent phosphate group.

•Complementary bases are perpendicular to the “backbone” and form the rungs of the ladder.

DNA replication is the synthesis of two DNA replication is the synthesis of two new DNA molecules from one that new DNA molecules from one that

already exists. already exists.

Replication

• Initiator protein binds at replication origin , separates the 2 strands & opens a replication bubble.

• Happens simultaneously at many points along molecule so replication occurs faster.

• DNA helicase attaches at the replication fork & unzips DNA molecule by breaking the hydrogen bonds between complementary bases.

• Each of the separated strands serve as templates. The templates make it possible to synthesize two daughter molecules of DNA identical to the original. Replication Fork

11stst step: Separation step: Separation

2nd Step: Polymerization

• RNA Primase binds to the separated strands of DNA, makes RNA “primer”.

• DNA Polymerase III binds

at primer, “reads” each nucleotide of old strand.

• Adds complementary nucleotides from those

floating free in nucleoplasm. • Joins adjacent nucleotides to

form the sugar-phosphate backbone.

• Creates two identical copies of original molecule. Each copy has one chain from original DNA molecule and one new chain of nucleotides.

3’ and 5’ Ends• Each DNA strand begins with the 5’ end of a

phosphate group on the first nucleotide and ends with the 3’ end phosphate group on the final nucleotide.

• DNA Polymerase III reads the strand in the 3’ to 5’ direction only and builds the new strand from 5’ to 3’ direction only.

•The strands run antiparallel to each other. Therefore one strand runs 5’ to 3’ and the other runs 3’ to 5’.

Leading and Lagging Strands

On each side of the replication fork there is a

different type of strand used for replication.

•DNA polymerase III reads 3’5’.•One side is the leading strand, which DNA polymerase reads continuously & synthesizes toward the replication fork. •The opposite side is the lagging strand, which runs in the opposite direction, 5’3’. •RNA primase reads the DNA, adding RNA in short segments. This is more complicated because DNA can not be read in the 5’ to 3’ direction. •DNA Polymerase III lengthens these segments, forming Okazaki fragments. •DNA Polymerase I then removes the RNA and adds its own nucleotides. •DNA ligase joins the fragments together.

Putting It All Together• http://www.bioteach.ubc.ca/TeachingResources/Molecul

arBiology/DNAReplication.swf• http://www.johnkyrk.com/DNAreplication.html• http://video.google.com/videosearch?hl=en&q=replicatio

n+animation&um=1&ie=UTF-8&ei=jCScSeSbHOH8tge0mITqBA&sa=X&oi=video_result_group&resnum=5&ct=title#

Troubleshooting• As replication bubbles open, DNA As replication bubbles open, DNA

twists begin to bind up.twists begin to bind up.

• An enzyme called An enzyme called topoisomerase topoisomerase cuts one or both strands of coiled cuts one or both strands of coiled DNA, making replication and DNA, making replication and transcription easier. transcription easier.

• http://video.google.com/videosearch?hl=en&q=replication+animation&um=1&ie=UTF-8&ei=jCScSeSbHOH8tge0mITqBA&sa=X&oi=video_result_group&resnum=5&ct=title#

Error and Mutation•Incorrectly paired nucleotides are a Mutation. (addition, deletion, substitution)•DNA polymerase III proofreads its work and usually removes & replaces incorrect nucleotides, and continues building the new strand. •Repair enzymes fix damage to the DNA molecule directly or with the help of DNA polymerase III &ligase.

•Hundreds of mutations are inherited from parents, but most mutations have little or no effect.

TranscriptionTranscriptionTranscription is the process in which genetic information is

copied from DNA to RNA.

The Need

• Cells are like little factories that make proteins.

• DNA codes for proteins.

• DNA remains in the cell’s nucleus, but proteins are made in the cytoplasm.

• mRNA is a copy of a gene that is sent into the cytoplasm to be “translated” into a protein.

The Process• Transcription is very similar to the

process of DNA replication.• RNA polymerase initiates

transcription by binding to the promoter region, (TATA box or CAAT box) just “upstream” of gene to be copied.

• Separates one twist (10 bp) of the DNA molecule.

• Reads 3’5’, builds mRNA 5’3’• Only reads the sense strand

(other one is the nonsense strand.)

Template

RNA Polymerase

The Process, Continued•RNA polymerase adds complementary RNA nucleotides to the newly forming RNA molecule one at a time.

•This continues until the polymerase reaches the termination signal, (a specific nucleotide sequence or a protein).

•RNA polymerase then releases the DNA molecule and the new RNA molecule.

•DNA retwists.

Try it!Transcribe the following sense strand:

TAC/GGA/CGT/GTT/CCC/GCG/ATG/ACC/CTA/GAT/TGA

AUG/CCU/GCA/CAA/GGG/CGC/UAC/UGG/GAU/CUA/ACU

Post-processing•In raw mRNA, sequences that do not code for anything, are called introns. They will be removed. •Sequences that do code for something, exons, are now next to each other and will be expressed as proteins.•A cap is added to the 5’ end - necessary for mRNA to bind to ribosome.•A poly-A tail is added to the 3’ end – helps mRNA move from nucleus to cytoplasm•Both prevent degradation of the molecule.

Structure of RNA• RNA is a single-stranded nucleic acid

that has a similar structure to that of DNA (chain of nucleotides attached by the sugar and phosphate groups). However, RNA’s sugar is ribose.

• The four nucleotide bases are adenine, guanine, cytosine, and uracil. In RNA, uracil replaces thymine (a DNA nucleotide).

• The same base pairing rules apply for RNA, except now uracil pairs with adenine.

A=Adenine U=UracilC=Cytosine G=Guanine

RNA MoleculesThere are three different types of RNA

molecules that are created during transcription

•Messenger RNA (mRNA): copy of a gene, carries the genetic information out of the nucleus into the cytoplasm for protein syntheses. It is a straight, uncoiled strand.

•Transfer RNA (tRNA): picks up amino acids in the cytoplasm & transfers them to the correct location in the growing peptide chain. It has a hairpin structure.

•Ribosomal RNA (rRNA): combines with proteins in the cytoplasm to create ribosomes. It has a globular structure.

Accuracy of TranscriptionAccuracy of Transcription• The RNA polymerase proofreads its work to

ensure accuracy.• As the enzyme is adding nucleotides it can also

remove them if a pair forms that is not complementary.

• RNA polymerase stays around the mismatched nucleotides longer, allowing time to remove the incorrect nucleotide.

• Error rate is just 1 in10,000 nucleotides added.

TranslationTranslation is the process of putting together polypeptides from

information encoded in mRNA.

Codons• Codon – 3-base sequence on mRNA that codes

for an amino acid• start codon: AUG, also codes for methionine. (It

tells ribosome where to begin translation, but that initial methionine is often removed later on in the process)

• stop codon (UAA, UAG, or UGA) - tell ribosome to stop translating the mRNA molecule.

• only 20 amino acids, so some amino acid have more than one codon.

tRNA • Transports amino acids to the

ribosomes.• The structure of tRNA is very

important to its job in translation. Each tRNA has an anticodon, a region which is complimentary to its corresponding codon on the mRNA molecule.

• A specific amino acid attaches on the opposite side of the molecule from the anticodon.

• This structure ensures that the amino acids placed precisely to be added to the polypeptide chain (protein).

Step One – Initiation

• The ribosome recognizes the start codon and attaches to it, holding the mRNA in place for easier translation.

• A special initiator tRNA molecule, carrying methionine and with anticodon UAC (complimentary to AUG) bonds to the start codon mRNA.

• A ribosome covers two codons simultaneously. Once the first two sites are filled, protein synthesis can continue.

Step Two - Elongation• The amino acid on the next tRNA attaches to the previous

amino acid by a peptide bond (covalent), forming a polypeptide chain, one amino acid at a time.

• As the ribosome moves on the mRNA, the first tRNA is released from the ribosome. However, the amino acid stays attached to the polypeptide chain, attached to the next tRNA.

• The movement of the ribosome is called Translocation. The ribosome moves (reads) in the 5’ to 3’ direction.

• As the ribosome moves it exposes new codons that attract the corresponding tRNA molecule with its amino acid.

• Ribosomes translate mRNA constantly, with one right after another.

http://bioweb.uwlax.edu/GenWeb/Molecular/Theory/Translation/translation.htmhttp://www.youtube.com/watch?v=41_Ne5mS2lstranslation

Step Three - Termination

• Elongation continues until the ribosome is at the site of the stop codon.

• The tRNA molecule and the polypeptide are released.

• The polypeptide will be used and processed in different parts of the cell depending on what it is suppose to do and its destination. Its function depends on the polypeptide sequence.

What’s the story?

• Only 10% of human genome codes for proteins (mRNA).

• Remaining 90%:

• Introns• Code for making rRNA & tRNA• Regulatory or control sequences (enhancers, promoters)• Repetitive sequences – function unknown (leftovers from

evolution?)

Proteins, a.k.a. Polypeptides

Protein diversity: variation in proteins is due to:

1. length of peptide chain (# of AA).

2. sequence of AA within the chain (order of AA).

3. 3-D configurations (shapes) due to folding of molecule.

4. protein may contain a single chain or multiple chains.

Amino Acids

• 20 Different AA’s (we make 12, must get 8 in diet).

 

Structure:

• “Backbone” is the same for each:

(amino group) R (carboxyl group)

H2N – C – COOH

H • Sidechain (R-group) differs for each AA, determines its

characteristics.

Protein Structure

• Bonding of AA’s – peptide (covalent) bonds form between carboxyl group of one AA and amino group of another.

R R R

H2N – C – COOH H2N – C – COOH H2N – C – COOH

H H H

• “Backbone” consists of the repeating sequence :

-N-C-C-N-C-C-N-C-C-N-C-C- plus all other atoms EXCEPT the R groups.

ProteinsProteinsProteins have four different Proteins have four different

levels of organization:levels of organization:• PrimaryPrimary (original): A long, (original): A long,

linear sequence of amino linear sequence of amino acids (string of beads). acids (string of beads).

• SecondarySecondary: results from : results from hydrogen bonding involving hydrogen bonding involving the backbone (N of amino the backbone (N of amino group with O of carboxyl group with O of carboxyl group).group).

1.1. Can form alpha-helix or Can form alpha-helix or beta-pleated sheet.beta-pleated sheet.

2.2. Like the coils of a telephone Like the coils of a telephone cord.cord.

• TertiaryTertiary: Folded coiled : Folded coiled (secondary) amino acid (secondary) amino acid chains. chains.

• QuaternaryQuaternary: Multiple amino : Multiple amino acid chains. acid chains.

Sidechain Interactions• Hydrophobic (non-polar) – orient themselves

toward the interior of the protein molecule.• Hydrophilic (polar) – orient toward the outside,

hydrogen-bond with water in cytoplasm.• Acidic (negative charge) attracted to basic

(positive charge), +/- pairing stabilizes protein.• Cysteine sidechains pair inside the molecule

to form a covalent disulfide bond (also stabilizes protein).

• Proteins assume the shape that gives them their maximum stability (lowest energy state).

Gene Expression

• Activation of a gene results in the expression of that gene by production of the protein(s) for which it codes.

• Repression ceases the production of proteins coded in a gene.

• Regulating gene expression saves resources and controls all cellular activity.

Gene Expression in Prokaryotes

• Operon – series of genes that code for specific products AND regulatory elements that control these genes.

Elements of an Operon• Structural genes – code for particular polypeptides.• Promoter – DNA segment that recognizes RNA

polymerase & promotes transcription.• Operator – DNA segment, serves as a binding site for an

inhibitory protein blocking transcription.

lac operon turned OFF

• When lactose is absent, a repressor protein (coded for by regulator gene) binds to operator & blocks attachment of RNA polymerase to structural genes.

lac operon turned ON

• When lactose is present, it acts as an inducer (binds to repressor proteins, preventing them from attaching to the operator, & RNA polymerase transcribes the structural genes).

• This is feedback inhibition.

Gene Expression in Eukaryotes

• Enhancer – noncoding control sequence on DNA.

• Transcription factors – proteins that bind to both enhancer & RNA polymerase to facilitate transcription.

Cell Differentiation

• Homeotic genes – regulatory genes determine WHERE certain structures will develop.

• Homeobox – sequences within homeotic gene for specific body regions.

Bibliographyhttp://www.rcsb.org/pdb/molecules/pdb40_1.htmlhttp://www.dnaftb.org/dnaftb/http://en.wikipedia.org/wiki/DNAhttp://www.answers.com/main/reference.jsphttp://www.mansfield.ohio-state.edu/~sabedon/biol1060.htmwww.andrew.cmu.edu/.../DNA_Replication.html http://www.alumni.ca/~mcgo4s0/t3/RNA.htmlhttp://www.rothamsted.ac.uk/notebook/index.htmlhttp://ull.chemistry.uakron.edu/biochem/12/http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/T/Translation.htmlhttp://www.johnkyrk.com/DNAtranslation.htmlhttp://www.medterms.com/script/main/art.asp?articlekey=32631

Travers, Bridget. The Gale Encyclopedia of Science. Volume 2.Encyclopedia of Life Sciences. Second Edition. Volume 4.