DNA: Chapters 16-18, 20

1. DNA: Chapters 16-18, 20 Structure Choose a topic: Replication Mutations Transcription/Translation Gene Expression Other Technologies Sources

2. Structure • Double helix made up of 4 bases; 2 purines that are made up of 2 rings (adenine, guanine), and 2 pyrimidines that are made up of 1 ring (thymine, cytosine) • A-T with 2 hydrogen bonds, G-C with 3 hydrogen bonds • Double helix has 10 nucleotide (NT) pairs per twist and keeps a constant diameter of 2nm • DNA always built in the 5’-3’ direction with the phosphate attached to the 5’ carbon of the sugar on one NT binding to the 3’ carbon of the sugar of the next NT • Makes up the sugar phosphate backbone phosphate sugar base Home

3. Packaging • DNA double helix has a diameter of 2nm • Wrapped around histones, make up nucleosomes referred to as “beads on a string”. Amino end of histone extends outwards (histone tail) - 10nm • The protruding histone tails interact and link together causing the 10nm to coil and fold condensing to 30nm, common during interphase • The 30nm fiber will loop into looped domains attached to a “protein scaffold” and condenses to a diameter of 300nm • Looped domains further coil into a metaphase chromosome 700nm in diameter • During interphase chromatin still sometimes condensed into heterochromatin vs. the less condensed euchromatin Home

4. Replication • DNA replication is guided by enzymes that are grouped together in a replisome/DNA replication complex • Steps of DNA replication • Replication starts at specific origins of replication, marked by specific sequences. Helicase opens the double helix, separating the parent strands, single strand binding proteins temporarily prevent repairing of base pairs • Primase creates a short sequence of 5-10 RNA nucleotides (NT) called a primer. DNA polymerase will build 5’-3’ • DNA polymerases require a primer and a template strand • Complementary bases added to the template (parent) strand, elongating the primer. NTs come from nucleotide triphosphates that lose phosphates when added, release energy to drive the process • Elongating towards the fork, polymerases added continuously, adding to the leading strand that will require only 1 primer, opposite direction (lagging strand) must keep restarting with multiple primers, make up Okazaki Fragments 100-200 NTs long that are formed by DNA Polymerase III, they are later joined together by DNA Ligase and RNA primers are replaced with DNA nucleotides by DNA polymerase I Home

5. After Replication • DNA polymerases check for errors, if this “proofread” misses errors, enzymes will swap in correct bases in process called mismatch repair before the errors become permanent mutations • Still damaged segments cut out by nuclease and filled back in with correct bases by polymerases using the template strand • Because strand only elongates 5’-3’, no way to repair the 5’ end, buffered by telomeres, sequences repeated 100-1000 times that become shorter as cells divide but protect coding sections. • Degeneration of telomeres contributes to aging, telomerase in germ cells continually elongates telomeres so that gametes will not eventually be without genes • Telomerase found to contribute to cancer, active in tumors allowing uninhibited division Home

6. Mutations • Point Mutation=changes in single NT pairs, can have small or large impacts • 3 types of point mutations: • Subsitutions = replacement of single NT pair with another pair • Insertions/deletions = +/- of NT pairs • Frameshift mutations alter the reading frame and thus change all subsequent codons • 3 categories of results from a mutation: • Silent Mutations have no effect on phenotype • Missense Mutations change what amino acid the codon codes for • Nonsense Mutations change the codon to a stop codon resulting in premature termination of the polypeptide chain • Common causes of mutations are: • X Rays • UV Radiation can cause thymines to bind to each other causing the DNA to buckle - thymine dimers are permanent damage Home

7. Transcription • Transcription = synthesis of RNA from DNA, RNA for a protein coding gene = messenger RNA (mRNA) • Yields primary transcript (pre-mRNA) in eukaryotes that needs processing before being usable • Prokaryotes primary transcript requires no modification before translation • RNA Polymerase can work without a primer, attaches at a promoter (includes start point for transcription) and stops at a terminator, stretch of DNA being transcribed is the transcription unit • Initiation: • In prokaryotes, RNA polymerase binds by itself, in eukaryotes, it will not bind until transcription factors have bound to the promoter • Polymerase + transcription factors = transcription initiation complex • TATA box = sequence commonly found in eukaryotic promoters • Elongation: • RNA polymerase moves down the template strand untwisting 10-20 NT at a time, building 5’-3’, 40 bases/sec in eukaryotes • Termination: • Prokaryotes: transcription proceeds through a terminator sequence that tells polymerase to detach and the strand needs no further modification • Eukaryotes: RNA Polymerase II transcribes a polyadenylation signal sequence that codes for a polyadenylation signal in pre-mRNA signaling proteins to cut it free 10-35 NT later. The strand needs further processing Home

8. RNA Processing • During RNA Processing, both ends of the primary transcript are altered, interior sections are sometimes excised • Alteration of ends: • 5’ end gets 5’ cap - modified guanine added after first 20-40 NT are transcribed • 3’ gets a poly-A tail - 50-250 adenine NT added to the 3’ end after the polyadenylation signal • Both: -facilitate export of finished mRNA from the nucleus -protect mRNA from degradation by hydrolytic enzymes -help ribosomes attach to the 5’ end once mRNA reaches the cytoplasm • Untranslated regions (UTRs) do not code for proteins, have other functions like ribosome bonding, on both the 5’ and 3’ ends • RNA Splicing = removal of large portions of an RNA molecule (primary transcript) • Most eukaryotic genes have large non-coding regions between coding regions. Non-coding regions (introns) are cut out while coding regions (exons) are expressed • RNA Polymerase II transcribes both introns/exons, directed to splicing sites directed by small nuclear ribonucleoproteins (snRNPs) that recognize splice sites from the ends of introns • snRNPs + proteins = spliceosome that cuts out introns, joins exons, catalyzes the process • Ribozymes = RNA molecules functioning as enzymes. RNA can function as an enzyme because of: single strand, specifid 3-D structure, some bases have functional groups that can act as catalysts, can H-bond with other nucleic acids • Some genes code multiple polypeptides depending on what sections of the primary transcript are treated as exons - alternative RNA splicing • Introns may help evolution as increased spacing between exons increases the likelihood of crossing over during meiosis Home

9. Translation • Transfer RNA (tRNA) transfers amino acids from the cytoplasm to the growing polypeptide chain in a ribosome • tRNA tranlates mRNA using anticodons - complimetary sequences to the mRNA • Aminoacyl-tRNA syntetases bind 1 specific amino acid (20 types of syntetases) to appropriate tRNA, tRNA with an amino acid attached = aminoacyl-tRNA (charged) • Some tRNAs can bond with multiple codons - wobble • Ex. U at the end of 5’ on an anticodon can pair with A or G in 3’ codon. This is why most redundancies differ in the 3rd base of codons • Ribosomes = made up of 3 sites: A (arrival), P (growing chain), E (exit). Large and small subunits • 1/3 proteins, 2/3 rRNA - most abundant form of RNA • Initiation: • Small ribosomal subunit binds to mRNA with initiator tRNA (Met.) and various initiation factors. Collectively make up translation initiation complex, large subunit then binds • Elongation: • Amino acids added to c-terminus end of the preceding amino acid, divided into 3 steps: • Codon recognition - tRNA pairs with anticodon, GTP used for energy • Peptide bond formation - rRNA in large subunit catalyzes formation of peptide bonds between amino group of new amino acid in the A site to the carboxyl end of the polypeptide in the P site • Translocation - GTP used to move A siteP site, P siteE site • Multiple ribosomes can translate one mRNA at once - polyribosome • Termination: • Elongation stops when stop codon reaches A site, release factor binds to the stop codon in the A site, adds water molecule that cuts the polypeptide chain out of the P site Home

10. After Translation • Proteins often fold on their own because of primary structure, sometimes aided by chaperonin • Amino acids may have sugars/lipids/phosphates added • Enzymes may remove amino acids form the amino (leading) end • Chain may be cut into multiple pieces • Signal polypeptides signal the ribosome making the polypeptide to either move to the rough ER or stay in the cytoplasm Home

11. Gene Expression • Prokaryotes regulate gene expression through regulating transcription • Operons = operator (on/off switch)+promoter+genes controlled • Operons can be repressible (on unless turned off by a repressor protein) or inducible (off unless turned on by an inducer) • Eukaryotes don’t use operons • More condensed DNA sections (heterochromatin) less expressed • Histone acetylation promotes transcrption, methylation of bases (usually cytosine) does the opposite • Control elements = non-coding segments that serve as transcription factor binding sites • Transcription factors = activators/repressors for enhancers and mediator proteins that help enhancers/promoters interact when far away • Transcription can also be regulated through initiator proteins • Lifespan of mRNA also contributes to gene expression • Duration of gene’s expression can by regulated by Ubiquitin which binds to proteins to signal proteasomes to degrade them Home

12. Other Technologies • Gene cloning can be used to amplify a gene or produce a protein product ex. insulin • Plasmids used as DNA vectors to get target genes into host cell. Restriction enzymes used to excise target genes, fragments = restriction fragments that are connected into the plasmid by DNA Ligase • Polymerase Chain Reaction can quickly replicate specific DNA sequences in a test tube • Gel electrophoresis can determine fragment length, smaller ones go further in gel • Southern Blotting allows for detection of bands of a specific fragment in electrophoresed gel • First form of DNA sequencing = chain termination - looking at the last base added to determine overall sequence • Now done as sequencing by synthesis - each base added is detected • Invitro mutagenesis = figuring out what certain genes do by turning them off • RNA interference also finds gene function but by blocking translation or breaking down it’s mRNA Home

13. Sources Textbook Class Notes Images: http://www.chemguide.co.uk/organicprops/aminoacids/dnachain2.gif http://www.2classnotes.com/images/12/science/biology/botany/packaging_dna/eukaryoti_chromosome.gif http://mendel.informatics.indiana.edu/~samdchap/Project/Pictures/alternative_splicing.gif http://biology.kenyon.edu/courses/biol114/Chap05/trna-1.gif http://upload.wikimedia.org/wikipedia/commons/e/e9/Transcription.jpg http://metamodern.com/b/wp-content/uploads/2009/07/ribosome_diagram.gif http://2.bp.blogspot.com/_bDXG-tBJ9qU/TJvtBXcfr4I/AAAAAAAAAAM/CotAN2KXooA/s1600/DNA+replication.jpg Home