From Gene to Protein

1. From Gene to Protein Chapter 17

2. What Is a Gene? The idea of the gene has evolved through the history of genetics We have considered a gene as A discrete unit of inheritance A region of specific nucleotide sequence in a chromosome A DNA sequence that codes for a specific polypeptide chain

3. Proteins= link between genotype and phenotype

4. Flow of Genetic Information • DNA= “directions” are encoded in nucleotide sequences • Sequences provide information for protein synthesis • Gene expression= process by which DNA directs protein synthesis • 2 stages: transcription and translation

5. Flow of Genetic Information • Central Dogma • Concept that cells are governed by a cellular chain of command: DNA RNA protein • RNA= link between genes and the proteins • Transcription= synthesis of RNA under the direction of DNA • messenger RNA (mRNA) • Translation= synthesis of a polypeptide, using information in the mRNA

6. Flow of Genetic Information DNA RNA Protein

7. Nuclearenvelope • Bacteria • Translation can begin before transcription finishes mRNA molecule • Eukaryotes • Transcription occurs in nucleus • Translation occurs in cytoplasm DNA TRANSCRIPTION Pre-mRNA RNA PROCESSING mRNA DNA TRANSCRIPTION mRNA Ribosome TRANSLATION Ribosome TRANSLATION Polypeptide Polypeptide (a) Bacterial cell (b) Eukaryotic cell

8. Variation in Transcription and Translation • Bacteria and eukaryotes differ in.. • RNA polymerases • Termination of transcription • Ribosomes • Archaea are prokaryotes, but share many features of gene expression with eukaryotes

9. Form = Function • High diversity of molecules due to arrangement of monomers • DNA composed of 4 nucleotides • RNA composed of 4 nucleotides • 20 amino acids (monomers of proteins)

10. Form = Function • The flow of information from gene to protein is based on a triplet code • Nonoverlapping, three-nucleotide codon • Codons of a gene are… • Transcribed into complementary codons of mRNA • Translated into amino acids

11. Second mRNA base A U C G UUU UAU UCU UGU U Phe Cys Tyr UUC UCC UAC UGC C U Ser UUA UCA UGA Stop A UAA Stop Leu Trp UUG UCG UGG G UAG Stop CUU CCU U CAU CGU His CUC CAC CGC C CCC C Leu Pro Arg CUA CCA CGA A CAA Gln CUG CCG CGG G CAG First mRNA base (5 end of codon) Third mRNA base (3 end of codon) AUU AAU ACU AGU U Ser Asn C AUC Ile AAC ACC AGC A Thr AUA AAA ACA AGA A Lys Arg Met orstart AUG ACG AGG AAG G GUU GCU GAU GGU U Asp GUC GCC C GGC GAC G Val Ala Gly Gly GUA GCA GGA A GAA Glu GUG GCG GGG G GAG

12. Cracking the Code All 64 codons were deciphered by the mid-1960s Of the 64 triplets 61 codons represent amino acids 3 codons are “stop” signals Genetic code Redundant 1 amino acid coded by multiple codons Not Ambiguous 1 codon = 1 amino acid

13. Cracking the Code • Codons must be read in the correct reading frame • 5’ to 3’ direction • 5’-AUGCUGGAACCGACCUGA-3’

14. Evolution of the Genetic Code Genetic code is nearly universal… From unicellular organisms…. ..to multicellularanimals

15. Evolution of the Genetic Code Genes can be transcribed and translated after being transplanted from one species to another Tobacco plant expressing firefly gene Pig expressing jellyfish gene

16. Green sea slug expresses algal DNA • Green sea slugs (Elysia chlorotica) have functional chloroplasts that carry out photosynthesis • Chloroplasts are taken up from food source= algae • Algae DNA incorporated into slug DNA

17. Transcription DNA • Occurs in Nucleus • RNA made from DNA directions • Messenger RNA (mRNA) • Only one strand of DNA transcribed • Template strand= 3’ to 5’ • RNA made in 5’ to 3’ direction RNA

18. Transcription DNA • Enzyme= RNA polymerase • Purpose of RNA polymerase • Separates DNA strands • Links RNA nucleotides together • Several dozen nucleotide pairs in length • Section of DNA transcribed = transcription unit RNA

19. Transcription DNA • mRNA= complementary to DNA template strand • same base-pairing rules as DNA, with one exception • Uracil substitutes for thymine • Uracil-Adenine • Eukaryotes • Primary transcript= Initial RNA molecule produced by transcription • Additional processing needed to make final mRNA RNA

20. Transcription: Initiation • Promoter= nucleotide sequence signals binding site for RNA polymerase • Transcription initiation complex= RNA polymerase II + transcription factors • Transcription factors= proteins control translation of a gene by activating or preventing binding of RNA polymerase to promoter site • A promoter called a TATA box is crucial in forming the initiation complex in eukaryotes

21. 1 3 2 A eukaryotic promoter Figure 17.8 Promoter Nontemplate strand DNA 5 3 T A A A A A T 5 3 A T A T T T T TATA box Template strand Start point Several transcriptionfactors bind to DNA Transcriptionfactors 5 3 3 5 Transcription initiationcomplex forms RNA polymerase II Transcription factors 5 3 3 5 3 5 RNA transcript Transcription initiation complex

22. Transcription: Elongation RNA polymerase untwists the double helix 10-20 bases at a time Eukaryotes Rate of Transcription= 40 nucleotides/second Nucleotides= added to the 3 end of the growing RNA molecule

23. Nontemplatestrand of DNA Figure 17.9 RNA nucleotides RNApolymerase C C A A T A 5 T 3 U T C end 3 G T U A G C A C C U C A A C A A 5 3 T A G G T T 5 Direction of transcription Templatestrand of DNA Newly madeRNA

24. Transcription: Termination • Prokaryotes • Terminator= nucleotide sequence signaling end of gene, RNA polymerase separates from DNA • mRNA does not need additional modification • Translation can begin immediately • Eukaryotes • Polyadenylation signal sequence= RNA transcript released 10–35 nucleotides past this sequence • Additional processing needed to convert transcript into functional mRNA molecule • Processing occurs before leaves nucleus

25. 2 3 1 Promoter Transcription unit Figure 17.7-4 5 3 3 5 DNA Start point RNA polymerase Initiation Nontemplate strand of DNA 3 5 5 3 Template strand of DNA RNAtranscript UnwoundDNA Elongation RewoundDNA 3 5 3 5 3 5 RNAtranscript Termination 3 5 5 3 3 5 Completed RNA transcript Direction of transcription (“downstream”)

26. RNA Processing • Enzymes in the eukaryotic nucleus modify RNA transcript before released to cytoplasm • Alteration of ends of the primary transcript • RNA splicing of interior portions of molecule

27. RNA Processing • Each end of a pre-mRNA molecule is modified in a particular way • 5 end= modified nucleotide 5 cap • 3 end= poly-A tail • Reasons for modifications • Facilitate export of mRNA from nucleus • Protect mRNA from enzymes in cytoplasm • Assist with ribosomal attachment to 5 end

28. RNA Processing • Most eukaryotic genes have introns between coding regions • Introns= series of nucleotides in noncoding regions of genes • Included in RNA transcripts • Exons= coding regions of genes • Expressed when translated into amino acid sequences • RNA splicing removes introns and joins exons • Spliceosomes • Ribozymes • End product= mRNA molecule with continuous coding sequence

29. What is the purpose of introns? Sequences may regulate gene expression Genes can encode more than one kind of polypeptide Type of polypeptide depends on which segments are removed during splicing Alternative RNA splicing Advantage= Number of different proteins produced is much greater than its number of genes

30. RNA Processing Spliceosomes= proteins + small nuclear ribonucleoproteins (snRNPs) • Recognize splice sites

31. RNA Processing Ribozymes= catalytic RNA molecules Function as enzymes Splice RNA Discovery changed long-held belief that all biological catalysts were proteins Three properties of RNA enable it to function as an enzyme Form a 3-D structure Base-pair with itself Bases contain functional groups that act as a catalyst Hydrogen-bond with other nucleic acid molecules

32. RNA Processing Exon Intron Exon Intron Exon 5 3 Poly-A tail Pre-mRNACodonnumbers Cap 5 130 31104 105 146 Introns cut out andexons spliced together Final Product 5 mRNA Cap Poly-A tail 1146 UTR 3 UTR 5 Codingsegment

33. Flow of Genetic Information DNA RNA Protein Transcription Translation

34. Transcription Translation • Flow of information from DNA to protein is the triplet code ofbases • mRNA moves out of nucleus and into cytoplasm • Ribosomes attach to mRNA and translation begins

35. Translation • Ribosomes “read” code on mRNA to build polypeptide chain • Amino acids brought to ribosome by transfer RNA (tRNA) • Single RNA strand • ~80 nucleotides long RNA Ribosomes Protein

36. Translation Molecules of tRNA pair with a specific amino acid • 3’ End= Amino acid attachment site • Anticodon • Base-pairs with complementary codon on mRNA • Hydrogen bonds give tRNA its 3-D structure • L-shaped

37. Translation • Accurate translation requires 2 steps: • Match between tRNA and an amino acid • Enzyme aminoacyl-tRNA synthetase • Match between tRNA anticodon and mRNA codon • Wobble= flexible pairing at the third base of a codon • Allows some tRNAs to bind to more than one codon

38. Aminoacyl-tRNAsynthetase (enzyme) Figure 17.16-4 Amino acid P Adenosine P P P Adenosine P P i Aminoacyl-tRNAsynthetase ATP P tRNA i P i tRNA Aminoacid P Adenosine AMP Computer model Aminoacyl tRNA(“charged tRNA”)

39. Ribosomes Facilitate specific coupling of tRNA anticodons with mRNA codons Two ribosomal subunits- large and small Proteins and ribosomal RNA (rRNA)

40. Ribosomes • Two types of ribosomes • Free ribosomes= in the cytosol • Bound ribosomes= attached to the endoplasmic reticulum (ER) • Both types are identical • Switch from free to bound • Free ribosomes mostly synthesize proteins that function in the cytosol • Bound ribosomes make proteins of the endomembrane system and proteins to be secreted from the cell

41. Ribosomes • Polypeptide synthesis always begins in the cytosol • Finishes in the cytosol unless the polypeptide signals the ribosome to attach to the ER

42. Ribosomes • Three binding sites for tRNA • P site= holds the tRNA with the growing polypeptide chain • A site= holds the tRNA with next amino acid to be added • E site= exit site

43. Figure 17.17c Growing polypeptide Amino end Next aminoacid to beadded topolypeptidechain E tRNA mRNA 3 Codons 5 (c) Schematic model with mRNA and tRNA

44. Translation: Initiation Brings together translation initiation complex: mRNA tRNA with first amino acid two ribosomal subunits Small ribosomal subunit binds with mRNA and initial tRNA Small subunit moves along the mRNA to start codon (AUG) Proteins called initiation factors bring in the large subunit

45. Translation: Initiation • First tRNA attaches at P site • All other tRNA enter at A site

46. Translation: Elongation Amino acids added one by one to the growing chain Proteins called elongation factors Occurs in three steps Codon recognition Peptide bond formation Translocation Translation proceeds along the mRNA in a 5′ to 3′ direction

47. Amino end ofpolypeptide Figure 17.19-4 E 3 mRNA Ribosome ready fornext aminoacyl tRNA Asite Psite 5 GTP GDP  P i E E P A A P GDP  P i GTP E A P

48. Translation: Termination • Stop codon in the mRNA reaches the A site of the ribosome • A site accepts a protein called a release factor • Release factor causes the addition of a water molecule instead of amino acid • Reaction releases polypeptide • Translation assembly separates

49. Translation Polyribosome: multiple ribosomes translate a single mRNA simultaneously Enable cell to make many copies of a polypeptide very quickly

50. Completedpolypeptide Growingpolypeptides Incomingribosomalsubunits Polyribosome Start ofmRNA(5 end) End ofmRNA(3 end) (a) Ribosomes mRNA (b) 0.1 m