DNA Replication and Protein Synthesis

1. DNA Replication and Protein Synthesis Taylor Reich January 8, 2010

2. Structure of DNA • Sugar+Phosphate+BasePair(A, G, C, or T)=Nucleotide(basic unit) • Purrines- A, G; Pyrimedines- C, T • A bonds with T- Double Hydrogen Bonded • G bonds with C- Triple Hydrogen Bonded • Covalent Bonds- sugar- phosphate, sugar-basepair • 3’ and 5’ ends • DNA -> hystome -> nucleosome -> supercoil • Major/Minor Grooves- hollow center (Think Staircase) • Two Thins for DNA • DNA Replication (self-replicates) • Protein Synthesis purines pyrimidines Sugar Phosphate

3. DNA Replication in Prokaryotes • Enzymes read opposite directions • 500 Nucleotides/second • Origin of Replication • Ring DNA

4. DNA Replication • Semi-conservative • Half old DNA, half new DNA • DNA polymerase (replicates DNA) • Reads 3’->5’; Makes 5’->3’

5. Eukaryotic Replication: Enzymes and their Functions • DNA B- mark replication site • DNA gyrase- uncoil DNA • Topoisomerase- knick DNA (release tension) • rep(helicase)- split DNA (break hydrogen bonds) • SSB (single strand binding)- hold complimentary DNA apart

6. Eukaryotic Replication: Enzymes and their Functions • RNA Primase- attach 10 RNA nucleotides • Okazaki fragments (lagging strand) • DNAPolymerase III- replicate okazaki fragments (lagging) DNA strand (leading) • DNA Polymerase I- replace RNA nucleotides with DNA ones (from primer) • Ligase- bond nicks from topoisomerase

7. Eukaryotic DNA Replication • 50 nucleotides/second • Leading and Lagging Strand

8. Protein Synthesis • Transcription • Make RNA from DNA • Nucleus • Translation • Make protein from RNA • Ribosome (cytoplasm)

9. Transcription [1] Splits DNA [2] Synthesizes RNA nucleotides [3] Re-bonds DNA • Parts of a gene: • RNA Polymerase makes RNA • Binds to promoter if it’s on, it moves to the start signal, synthesizes on RNA Start signal Stop Signal TATA TAC ORF (random sequence) Termination Sequence Promoter on/off switch • Open Reading Frame • codes for protein

10. Transcription • Have a mRNA strand from DNA • Add: • Cap: composed of 7 guanines; put on 5’ end • Tail: composed of 100-200 adenines; put on 3’ end • The more adenines attached, the longer the mRNA lasts • Remove: • SNRNP [Small Nuclear Ribo-Nuclear Protein] • Cuts out introns • Hooks together exons • [Spliceosome (SNRNPS&other proteins)] • Introns stay IN the nucleus; Exons EXIT the nucleus and continue on to the ribosome for translation…

11. Amino Acid (each tRNA has specific amino acid) Anticodon (three nucleotides) Translation • Ribosomes • Split/Come together all the time • Made up of two parts: small unit & large unit • tRNA (transfer RNA) • aminoacyl-tRNA synthetase • Whole group of slightly different enzymes. Each has 2 binding sites: 1) particular tRNA 2) particular amino acid

12. Translation • Step 1: small unit of ribosome binds with 5’ end of mRNA • Step 2: initiation factors(proteins) bind to mRNA and ribosome • Step 3: tRNA binds using initiating factors (on start signal of mRNA) (anitcodon binds) • Every start signal on mRNA is AUG (so tRNA nucleotide is UAC) • Step 4: Large unit of ribosome binds, leaving a space b/t the 2 parts of theribosome- the A-site, and the P-site • Step 5: knock off initiating factors, attach elongation factors- activate ribosome to do this: • Look for tRNA w/ completmentary anticodons to mRNA codon at the A-site • Step 6: peptidyl transferase forms a peptide bond b/t amino acids, and breaks bond between amino acid and tRNA at the P-site • Step 7: mRNA moves down 1 codon, tRNA at P-site, leaves, tRNA shifts, ribosome does it’s job, new tRNA arrives, whole process repeats • Result: A chain of Amino Acid, A.K.A. a PROTEIN

13. Transformation Tidbits • The sequence of nucleotides on mRNA determines the correct order of the amino acid chain, determining the correct protein…or not • Frame Shifting- loosing/gaining a nucleotide- changes every codon= wrong protein produced • Substitution- affects only one codon- not always a different amino acid • “Wobble Affect”- if the first or second letter (base pair) are wrong, it will be a different amino acid, if the third letter is wrong, it may not be • Release/Termination factor- binds to P-site at the end of the mRNA chain, ribosomes split • Can be more than one ribosome on a mRNA chain at a time • Polycistronic: a messenger RNA that codes for more than one protein

14. Stops the production Negative Feedback • When DNA is read, producing enzymes, creating a product, the product concentration gets high. Two things can happen: • Product binds to the DNA • Product binds to the enzymes • When the concentration gets low enough, the product falls off of the DNA/enzymes, commencing production once again • Operator- b/t promoter and start signal • Regulator- before promoter, usually turned on being read, giving out mRNA. It makes the… • Repressor- a protein that binds to the operator, turning off the gene Regulator Promoter Operator Start Signal ORF Termination Signal

15. Negative Feedback:Induction, Corepression, & CAP • Inducer- a substrate that binds to repressor, deactivating it/knocking it off the DNA. The DNA is turned on. When it falls off the repressor, the repressor sits on the operator, turning off gene. It is called Induction • Sometimes, the product is the substrate for the inducer, so when there is a high concentration of product, it turnes off the gene. That is called Corepression • The product concentration is the important thing in these processes • Overriding the system is possible with CAP. Turns on the gene when cAMP sits on DNA, the DNA bends, knocking off the repressor, activating CAP, and the gene turns on • cAMP goes to mitochondria, gets phosphates, turns to ATP, CAP comes off DNA, DNA goes back to original shape, repressor binds w/ corepresser, sites on DNA, and the gene turns off • Can’t be done consciously

16. More on Gene Structure About 10,000 base pairs gene • DNA bends so that the Initiation Gene touches the Promoter, which is bond to the Transcriptional Factor Gene, the Enhancer Gene, and the DNA