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From Gene to Protein

From Gene to Protein. Campbell and Reece Chapter 17. Gene Expression. process by which DNA directs the synthesis of proteins or RNA synthesis of proteins transcription translation. How Gene to Protein Figured Out. Evidence from study of metabolic disorders:

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From Gene to Protein

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  1. From Gene to Protein Campbell and Reece Chapter 17

  2. Gene Expression • process by which DNA directs the synthesis of proteins or RNA • synthesis of proteins • transcription • translation

  3. How Gene to Protein Figured Out • Evidence from study of metabolic disorders: • 1902: British physician 1st to suggest genes responsible for phenotype thru enzymes that catalyze specific chemrx in the cell

  4. Inborn Errors of Metabolism • Garrod hypothesized that symptoms of an inherited disease are due to a gene that leads to inability to make a certain enzyme • 1 of 1st to realize Mendel’s principle’s of heredity applied to more than pea plants

  5. Alkaptonuria • Signs & Symptoms: • urine turns black when alkapton (chemical in urine) reacts with air • missing enzyme in pathway that degrades phenylalanine (a.a.)

  6. Beadle & Tatum Experiment • worked with a bread mold Neurosporacrassa • bombarded it with radiation (already known to cause genetic changes) • then checked for survivors who had different nutritional needs from wild-type mold

  7. Beadle & Tatum Experiment • individually put yeast in different mediums (agar with different nutrients) • identified mutants that could not survive on minimal nutrients placed them in complete growth medium (minimal med. + all 20 a.a. + few vitamins & minerals)

  8. 1-Gene-1-Polypeptide • Beadle & Tatum’s results supported their hypothesis • 1958: Nobel prize

  9. 1-Gene-1-Polypeptide • revised over time: • not all proteins are enzymes • some proteins have >1 polypeptide • now: 1- gene-1-protein hypothesis • not 100%: some eukaryotic genes can each code for a set of closely related polypeptides via alternative splicing

  10. Transcription: short version • the synthesis of RNA using information in DNA • mRNA made using complimentary base pairing

  11. Translation: short version • synthesis of a polypeptide using the information in mRNA • “translates” message in mRNA  a.a.

  12. The Genetic Code • 4 nucleotide bases to code for 20 a.a. • triplet code: 3 consecutive bases code for 1 of the a.a./ stop

  13. Template Strand • during transcription: • DNA helix unwound • 1 strand only transcribed (could be either side depending on the gene)

  14. mRNA • uracil added as compliment to adenine • ribose as its 5-carbon sugar • single stranded

  15. Codons • nucleotide triplets of DNA or mRNA that specifies a particular amino acid or termination signal • basic unit of the genetic code • written in 5’  3’ direction (in DNA 3 bases read in 3’  5’ direction)

  16. Genetic Code

  17. Cracking the Code • early 1960’s • Nirenberg: synthesized mRNA using only uracil (UUUUUUU…) • added it to test tube with all 20 a.a., ribosomes • translated into polypeptide made up of phenyalanine • now knew UUU = Phe • did same for AAA= Lys, CCC = Pro, GGG = Gly

  18. Cracking the Code • all 64 a.a. deciphered by mid-1960’s • 3 codons code for “stop” marking end of translation • AUG functions as “start” & Met • Met may or may not be clipped off later

  19. Genetic Code is Redundant • >1 triplet codes for each of the a.a. but any 1 triplet codes for only 1 a.a • redundant triplets usually only differ in the 3rd base

  20. Reading Frame • translating the code in correct groupings • example: Did the red dog eat the bug? Idt her eddogeatthebug?

  21. Reading Frame

  22. Evolution of Genetic Code • code is nearly universal: • bacteria  complex multicellular organisms CAU = His • insert genes into other species & get same result (human insulin gene in bacteria) • exceptions: certain unicellular eukaryotes & in organelle genes of some species

  23. RNA Polymerase • unwinds 2 strands of DNA • binds nucleotides together as build mRNA • only in 5’  3’ direction (like DNA polymerase)

  24. 3 Stages of Transcription • Initiation • Elongation • Termination

  25. Initiation • After RNA polymerase binds to promoter, ¤ DNA strands unwind • polymerase begins RNA synthesis @ start pt. on template strand

  26. Initiation • promoter: usually includes w/in it the transcription start point (a nucleotide where transcription begins) & extends several dozen or more nucleotide pairs upstream from start pt. • RNAP can assemble nucleotides only in 5’  3’ direction (just like DNA polymerase) • unlike DNAP, RNAP does not require a primer

  27. Start Point • nucleotide where RNA synthesis actually begins • RNAP binds in precise location & orientation on the promoter  where determines where transcription starts & which of the 2 strands will be transcribed

  28. RNA Polymerase • Bacteria: • 1 single RNAP used to make all types RNA • Eukaryotic Cells: • @ least 3 types RNA polymerase • II used for RNA synthesis • I and III used to transcribe RNA not used for protein synthesis

  29. RNA Polymerase • Prokaryotes : • RNAP recognizes & binds to the promoter by itself • Eukayotes: • collection of proteins , transcription factors, mediate the binding of RNAP & initiation of transcription

  30. Transcription Factors • must 1st attach to promoter b/4 RNAP II can bind to it • RNAP II + transcription factors = Transcription Initiation Complex • TATA box: DNA sequence in eukaryotic promoters crucial in forming the transcription initiation complex

  31. Elongation • RNAP moves downstrean, unwinding the DNA & elongating the RNA transcript 5’  3’ • ~ 10 – 20 nucleotides exposed • in wake of transcription the 2 DNA strands spontaneously rewind • length of DNA transcribed = transcription unit

  32. Elongation

  33. Termination • mechanism differs between prokaryotes & eukaryotes • Bacteria: transcription proceeds thru terminator sequence in the DNA  the transcribed RNA functions as the terminator sequence  causing RNAP to detach • prokaryotes have no further modification

  34. Termination in Eukaryotes • RNAP II transcribes a portion of DNA called the polyadenylation signal (AAUAAA) in the pre-mRNA • ~10 – 35 nucleotides downstream from that sequence proteins ass’c with transcription cut the pre-mRNA free from the polymerase • pre-mRNA then  modified

  35. RNA Processing • in eukaryotes only • both ends of primary transcript altered • certain interior sections cut out & remaining parts spliced back together

  36. mRNA Ends • 5’ end receives a 5’cap: modified G is added after ~ 20 – 40 nucleotides in mRNA • 3’ end modified: enzyme adds 50 -250 A’s to the AAUAAA forming a poly-A tail

  37. Functions of Modified Ends of mRNA • facilitate exit of mRNA from nucleus • protect mRNA from degradation of hydrolytic enzymes • help ribosomes attach to the 5’ end

  38. RNA Splicing • cut-and-paste job removing segments of RNA that were transcribed • average size transcript: 27,000 nucleotides • average size protein: 1,200 nucleotides (400 a.a.)

  39. Introns • noncoding, intervening sequence w/in primary transcript that is removed from the transcript during RNA processing; also refers to the region of DNA from which this sequence was transcribed

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