DNA and PROTEIN SYNTHESIS “Cracking the Code”

DNA the blueprint of life contains the instructions for making proteins within the cell. Deoxyribonucleic Acid Found in the nucleus of the cell (mitochondria also have DNA)

The Shape of DNA very long polymer a ‘twisted ladder’ or zipper called a double helix.

Prokaryotic vs Eukaryotic DNA Prokaryotic- found free moving in the cytoplasm, not contained by a nucleus. DNA tends to be circular in plasmid. Eukaryotic – found contained inside of the nucleus, DNA tends to be linear, and uses histones to coil it tightly.

Structure of DNA A 5 carbon SUGAR (deoxyribose)& phosphate backbone rungs that connect the backbones are nitrogenous bases Nucleotide = sugar + phosphate + nitrogenous base This structure was discovered by Watson, Crick, Franklin, and Wilkins DNA structure

Structure of DNA Anti parallel: one strand runs 5’ to 3’, the other runs opposite

4 DNA Bases Purine base - double rings. Adenine Guanine Pyrimidine base -single ring Thymine Cytosine

Complementary Pairing complementary base pairs: normal pairing of nitrogenous bases is: 1 purine and 1 pyrimidine Adenine always and only binds with Thymine A-T Guanine always and only pairs with Cytosine G-C -These ratios was discovered by Erwin Chargoff

DNA Replication semi-conservative replication-new DNA molecule made of one parent and one newly replicated strand. (Meselson and Stahl) In general a DNA molecule ‘unzips’ down the middle of the paired bases, 2 individual strands are made that will become the ‘templates’ for new complete DNA stands

The Steps for DNA Replication (during S-phase of Interphase): Initiation starts at a specific necletide sequence, a group of enzymes called DNA helicases breaks hydrogen bonds between bases to unzip the double helix Proteins bind to keep strands apart

RNA primers attach to a spot on the original DNA stand DNA polymerase III – starts at where the primer attached to the DNA and makes new strand in 5’ to 3’ direction (always) -DNA polymerase III can only be added to a 3’ end

DNA polymerase 1 – removes primers and replaces with nucleotide DNA ligase – joins DNA fragments

DNA makes DNA

DNA Editing AMAZING!!! DNA has a spell check DNA polymerase cut out mismatched base, and replace it with the proper nucleotides.

Protein Synthesis Proteins -chains of amino acids. as small as 8 amino acids, and as large as over 50,000 amino acids. There are 20 amino acids One Gene One Protein Theory - production of each protein is controlled by one gene. (The sequence of nucleotides in a gene will determine the structure/type/role of the protein) This is the CENTRAL DOGMA of DNA that Watson described

(Protein Function) Functions: - Pigments – eg. Melanin – protect or signal etc.

(Protein Function) - enzymes – eg. Amylase – catalyze reactions

(Protein Function) - movement – eg. Actin in muscle

(Protein Function) carriers – eg. Hemoglobin (oxygen)

(Protein Function) channels – in membranes (eg. Porin)

(Protein Function) - receptors – cell recognition eg. CD4 receptor on WBC (AIDS)

Essential amino acids

Ribonucleic acid (RNA) -required for protein synthesis, 1. RNA is single stranded, DNA is double stranded 2. The 5 carbon sugar is ribose in RNA, deoxyribose in DNA 3. RNA uses the base uracil instead of thymine. Uracil binds with adenine. (A-U )

3 Types of RNA Messenger RNA (mRNA) 2. Transfer RNA (tRNA) takes DNA code to the ribosomes where proteins are made 2. Transfer RNA (tRNA) brings the appropriate amino acids from the cytoplasm to the ribosomes and strings them in order according to mRNA.

3. Ribosomal RNA (rRNA) not directly involved with protein synthesis makes the ribosomes in the nucleus

Protein Synthesis

Steps in Protein Synthesis Transcription At a start sequence of neucleotides, the double stranded DNA opens up (unzipped by helicases again) RNA polymerase attaches to the promoter and builds the single stranded mRNA Stops at the termination sequence of nucleotides and mRNA detaches mRNA makes its way to the ribosome for translation DNA-RNA

2. Translation-making the protein

tRNA brings amino acids from the cytoplasm to the ribosome mRNA attaches between the two subunits of a ribosome which “reads” the mRNA mRNA is a codon (represents a 3 nucleotide sequence from the DNA that it just read) tRNA brings amino acids from the cytoplasm to the ribosome tRNA has an anticodon for an amino acid ex) If mRNA codes UUU, t RNA anticodon is AAA and it will get the amino acid PHE

mRNA Codons 3 nitrogen bases that code for a specific amino acid –WHY 3?

Start codon – starts protein synthesis, AUG which codes for methionine Stop codons – ends protein synthesis, UAA, UAG, UGA Intron – non-coding sequences Other non-coding regions of DNA are: telomeres, regulators, stop codon. Exon – coding sequences that make proteins

Once translation is complete tRNA returns to the cytoplasm and mRNA is broken down.

Changes or additions to transcription Methylation: The adding of a -CH3 (methyl) group to a cytosine on some area of the DNA strand. This methyl group now prevents RNA polymerase from transcribing any region past the methyl blockage. Usually involved in cell specialization Poly (aaa) tail- At the end of transcription a long section adenines can be added to the 3’ end (‘Tail’) of the mRNA strand that adds stability to the mRNA strand. These adenines prevent degradation of the mRNA stand

Why Do Firefly’s Glow? What Makes a Firefly Glow?

DNA and Mutations inheritable changes Spontaneous or caused by mutagenic agents Ex) radiation, chemicals may be as simple as a single base pair

Basic Mutations: Substitution of nucleotide(s), usually mild mutation created (but can be serious like sickle cell anemia) Frameshift – changes the reading frame. A whole new sequence is read, usually leads to severe mutations. Frameshifts are caused by: a) Deletion of a nucleotide(s) b) Addition of extra nucleotide(s) Translocation of a gene-DNA fragment switches location, often between different chromosomes. This is a very serious mutations (usually fatal) BBC Education - AS Guru - Biology - Genes and Genetics - Genetic Code - Mutation

Silent mutations: A mutation in the intron regions (non-coding sequences of DNA) or a mutation that causes a nitrogen base to change but not cause any change in amino acid produced. Usually due to a substitution mutation Missense mutation: a mutation that changes the DNA sequence so that a different amino acid is coded. Usually due to a substitution mutation A Nonsense mutation: a mutation that results in a stop codon being produced which results in no amino acids after the stop codon from being produced. Usually lethal

Mutations in body cells often have little consequences compared to mutations of the germ cells (sperm or egg). A single mistake in the DNA of a sperm or egg cell would be repeated billions of times if that cell underwent fertilization to become a complete individual.

Transposons are “jumping genes”, DNA sequences that have the ability to move in / out of chromosomes, changing their location. May cause mutations or change amount of DNA http://www.dnalc.org/vshockwave/ac_ds_trans.dcr

Oncogenes and Cancer Cancer -uncontrolled cell division from a changed genetic code (uncontrolled mitosis) Evidence: 1. Cancerous cells often display nitrogen base substitutions 2. Many known mutagens, are also known to cause cancer

segments of chromosomes extracted from cancerous mice transformed normal mouse cells into cancerous cells. -genes called oncogenes cause cancer

Oncogenes and Cancer oncogenes are present in normal cells, and do not always result in cancer oncogenes must be transposed to another site on the chromosome to be activated

The Ames Test To test for potential mutagens bacteria Salmonella typhimurium - unable to make histidine after the bacteria is exposed to a potential mutagen, it is grown in a culture without histidine. If it survives it has been mutated!

Biotechnology

Biotechnology –biological systems used to produce a product. Genetic engineering –produces transgenic cells: foreign DNA inserted Recombinant DNA – DNA that comes from 2 or more sources.

Vector –an organism (bacteria or virus) that carries and leaves its genetic material in a host cell. The host then replicates the vector’s genetic material. Plasmid – is circular DNA found in bacteria. This is often used as a vector in DNA recombination.

The process of creating recombinant DNA involves 2 types of enzymes: Restriction Enzymes (aka restriction endonucleases) – (DNA scissors) cut the DNA strand at specific sites -palindromes- and often creates sticky ends

2. The now unpaired segments can be paired with complimentary nucleotides from a different strand of DNA 3. DNA ligase – is genetic glue that puts DNA strands back together. rDNA

Application Insulin used to be harvested from pigs, but some individuals did not tolerate it. we are now able to use bacteria to produce human insulin in large quantities. is tolerated much better by patients. This procedure was first marketed in Canada in 1983!

Other Aspects of Biotechnology Gene sequencing –determining the specific location and composition of specific genes. The Human Genome Project -1990 2000: mapped our 30 000 genes (3 billion nucleotides) NOVA Online | Cracking the Code of Life | Sequence for Yourself

RFLP – restriction fragment length polymorphism –cut DNA into pieces Gel Electrophoresis – separate the DNA pieces by size.

DNA fingerprinting identify an individual based on their unique genetic code. homologous segments of DNA are similar but contain unique patterns of nitrogen bases identify individuals involved in crimes, paternity

DNA Fingerprinting

Misc. Technology Polymerase chain reaction –making billions of copies of a piece of DNA- PCR Animation We now use Taq from Thermus aquaticus bacterium to do PCR as it is resistanct to denaturing during the application of heat during PCR Paternity Testing - Paternity Testing Gene Gun - Genegun1