Chapter 12: RNA and Protein Synthesis

1. Chapter 12:RNA and Protein Synthesis Gene Expression – How DNA affects Phenotype DNA  proteins  phenotype

2. 2 steps • DNA  mRNA • Translation • mRNA  protein

3. Fig. 17-3b-3 Nuclear envelope DNA TRANSCRIPTION Pre-mRNA RNA PROCESSING mRNA TRANSLATION Ribosome Polypeptide (b) Eukaryotic cell

4. Fig. 17-4 Gene 2 DNA molecule Gene 1 Gene 3 DNA template strand TRANSCRIPTION mRNA Codon TRANSLATION Protein Amino acid

5. RNA – ribonucleic acid • Single stranded • nucleotides • Ribose • Phosphate • AUCG • U = Uracil

6. RNA • Transcription (DNA template  mRNA) • 3 types • Ribosomal RNA (rRNA) • Transfer RNA (tRNA) • Messenger RNA (mRNA) • Made from DNA – DNA-dependent RNA polymerases • Make RNA from DNA in 5’ 3’ direction, DNA read 3’5’ • DNA template and new RNA are antiparallel

7. Upstream – toward 5’ of mRNA OR 3’ of DNA • Downstream – toward 3’ of RNA OR 5’ of DNA

8. Transcription • 1. RNA polymerase binds to DNA at Promoter • Promoter not transcribed • RNA polymerase passes promoter; begins transcribing DNA • No primer required • 2. RNA nucleotides added to 3’ end of RNA • 1st RNA (5’ end) keeps triphosphate • RNA nucleotides added lose 2 P and 3rd P becomes part of sugar-phosphate backbone • Last RNA nucleotide – exposed 3’ OH

9. Transcription • 3. Termination • Stop sequence at end of gene

10. Fig. 17-8 A eukaryotic promoter includes a TATA box 1 Promoter Template 5 3 3 5 TATA box Template DNA strand Start point Several transcription factors must bind to the DNA before RNA polymerase II can do so. 2 Transcription factors 5 3 3 5 Additional transcription factors bind to the DNA along with RNA polymerase II, forming the transcription initiation complex. 3 RNA polymerase II Transcription factors 3 5 5 5 3 RNA transcript Transcription initiation complex

11. Fig. 17-7a-4 Promoter Transcription unit 5 3 3 5 DNA Start point RNA polymerase Initiation 1 5 3 3 5 Template strand of DNA RNA transcript Unwound DNA Elongation 2 Rewound DNA 5 3 3 3 5 5 RNA transcript Termination 3 5 3 3 5 3 5 Completed RNA transcript

12. Fig. 17-7b Nontemplate strand of DNA Elongation RNA nucleotides RNA polymerase 3 3 end 5 Direction of transcription (“downstream”) 5 Template strand of DNA Newly made RNA

13. Resulting mRNA • Leader sequence – • Noncoding • Made because RNA polymerase starts transcription well upstream of coding sequence • Coding sequence – • Codes for proteins • Termination or stop codon • End of coding sequence • UAA, UGA, UAG • Don’t code for AA; specify end of protein • Followed by noncoding 3’ trailing sequences

14. Transcription

15. Posttranscriptional modification and processing • Precursor mRNA = original mRNA transcript (pre-mRNA) • Begins – RNA transcript is 20-30 nucleotides long • Enzymes add cap to 5’ end of mRNA • Need cap for eukaryotic ribosome to bind • May protect from degradation

16. Fig. 17-9 Polyadenylation signal Protein-coding segment 5 3 … G AAA AAA P P P AAUAAA 5 Cap Poly-A tail Start codon 5 UTR 3 UTR Stop codon

17. Polyadenylated (poly-A) tail gets added • 3’ end • When transcript complete, enzymes in nucleus recognize polyadenylation signal and cut mRNA at that site • 100-250 adenine nucleotides are added by enzymes to 3’ end • May help • Export mRNA from nucleus, fight degradation, make translation initiation more efficient

18. Take out noncoding sequences • Interrupted coding sequences = long sequences of bases in the protein-coding sequences of the gene that do not code for AA in the final protein product INTRONS (noncoding regions) • EXONS – (expressed sequences) – part of the protein-coding sequence • Introns are removed and splice exons together  continuous coding sequence

19. Small nuclear ribonucleoprotein complexes (snRNPs) – bind to introns and catalyze the excision and splicing reactions

20. Fig. 17-10 Exon Exon Intron 5 Exon Intron 3 Pre-mRNA Poly-A tail 5 Cap 146 31 104 1 105 30 Introns cut out and exons spliced together Coding segment mRNA 5 Cap Poly-A tail 1 146 5 UTR 3 UTR

21. Fig. 17-11-3 RNA transcript (pre-mRNA) 5 Exon 1 Intron Exon 2 Protein Other proteins snRNA snRNPs Spliceosome 5 Spliceosome components Cut-out intron mRNA 5 Exon 2 Exon 1

22. mRNA processing

23. Fig. 17-13 Amino acids Polypeptide tRNA with amino acid attached Ribosome Trp Phe Gly tRNA Anticodon Codons 5 3 mRNA

24. Translation: mRNA  AA (protein) • Codon – sequence of 3 consecutive bases in mRNA (triplet code); specify 1 AA • Transfer RNA (tRNA) – connect AA and mRNA; link with specific AA • Anticodon – sequence of 3 bases on tRNA; H bonds with mRNA codon by base-pairing rules

25. Aminoacyl-tRNA synthetase – enzyme that links amino acids to tRNAs • Make aminoacyl-tRNAs (can bind to mRNA)

26. Fig. 17-15-4 Aminoacyl-tRNA synthetase (enzyme) Amino acid P P P Adenosine ATP P Adenosine tRNA P P i Aminoacyl-tRNA synthetase P i P i tRNA P Adenosine AMP Computer model Aminoacyl-tRNA (“charged tRNA”)

27. Properties of tRNA • 1. anticodon – 3 base triplet complementary to mRNA codon • 2. must be recognized by specific aminoacyl-tRNA synthetase that adds correct AA • 3. must have region that serves as attachment site for specific AA specified by anticodon • 4. must be recognized by ribosomes

28. tRNA • Gets folded on itself (base-pairing within tRNA)  3+ loops (unpaired bases) • 1 of the loops has anticodon • 3’ end – AA binding site • Carboxyl of AA binds to OH tRNA at 3’ end, leaving amino group on AA to make peptide bond

29. Fig. 17-14a 3 Amino acid attachment site 5 Hydrogen bonds Anticodon (a) Two-dimensional structure

30. Translation – At Ribosomes • Ribosome • Made of 2 different subunits (proteins and ribosomal RNA) • rRNA – no transfer of info, has catalytic functions • Attach to 1 end of mRNA and travel along it, allowing tRNAs to attach in sequence to mRNA codons

31. Ribosomes • mRNA fits in groove between 2 subunits • Holds mRNA, aminoacyl tRNA and growing polypeptide chain • tRNAs attach to A and P binding sites • A site – new AA dock; AA form bond with polypeptide chain and tRNA moves to P site

32. Fig. 17-16b P site (Peptidyl-tRNA binding site) A site (Aminoacyl- tRNA binding site) E site (Exit site) E P A Large subunit mRNA binding site Small subunit (b) Schematic model showing binding sites Growing polypeptide Amino end Next amino acid to be added to polypeptide chain E tRNA mRNA 3 Codons 5 (c) Schematic model with mRNA and tRNA

33. 3 stages of Translation • Initiation, repeating cycles of elongation, termination

34. Initiation • Initiation factors (proteins) attach to small subunit, allowing it to bind to a special initiator tRNA • Initiator tRNA is loaded onto small subunit, making initiation complex • Initiation complex binds to special ribosome-recognition sequences upstream of coding sequences on mRNA (near 5’ end) • Initiation complex moves along mRNA until it reaches an initiator codon = AUG

35. Anticodon of initiator tRNA binds to initiation codon of mRNA • Large subunit attaches to complex •  completed ribosome

36. Fig. 17-17 Large ribosomal subunit 3 U 5 C A P site Met Met 5 3 A G U Initiator tRNA GDP GTP E A mRNA 5 5 3 3 Start codon Small ribosomal subunit Translation initiation complex mRNA binding site

37. Elongation • Addition of AA to A site by base pairing of anticodon w/ codon • Ribosome moves in 3’ direction along mRNA • Needs energy from GTP • Peptidyl transferase – ribozyme – rRNA component of large subunit • AA at P site released from its tRNA (in P site) • Peptidyl transferase attaches this AA to aminoacyl-tRNA at A site • Peptide bond formed – translocation – chain moves to P site, leaving A site open • GTP for bond, none for transferase

38. Fig. 17-18-4 Amino end of polypeptide E 3 mRNA P site A site Ribosome ready for next aminoacyl tRNA 5 GTP GDP E E P A A P GDP GTP E P A

39. Termination • “Release factors” stop translation • Recognize termination (stop) codons • Release newly-made protein, mRNA and last tRNA, causing ribosome to dissociate

40. Fig. 17-19-3 Release factor Free polypeptide 5 3 3 3 2 GTP 5 5 Stop codon (UAG, UAA, or UGA) 2 GDP

41. Translation

42. Protein Synthesis:Eukaryotes vs. Prokaryotes Prokaryotes Eukaryotes mRNA must be transported to cytoplasm before translation Original mRNA transcript must be modified before leaving the nucleus • mRNA is translated as it is being transcribed from DNA (no nucleus to exit) • mRNA used immediately, no further processing

43. Fig. 17-3 DNA TRANSCRIPTION mRNA Ribosome TRANSLATION Polypeptide (a) Bacterial cell Nuclear envelope DNA TRANSCRIPTION Pre-mRNA RNA PROCESSING mRNA TRANSLATION Ribosome Polypeptide (b) Eukaryotic cell

44. Special features of the Genetic Code • 3 letter combos from 4 bases – specify 64 AA • Nirenberg and Matthaei • Experimented to determine which AA were coded for by specific mRNA codons • Ex: UUUUUUUUU… - only found phenylalanine so UUU = phenylalanine • Found 3 stop codons – specified no AA • UAA, UGA, UAG

45. Fig. 17-5 Second mRNA base First mRNA base (5 end of codon) Third mRNA base (3 end of codon)

46. The Genetic Code is Universal • All organisms • Few coding exceptions • Protozoans – UAA, UGA for glutamine, instead of stop • Mitochondria - own DNA

47. Wobble Hypothesis • 61 codons, but only 40 different tRNAs •  tRNA can pair w/ 1+ codon • Francis Crick • 3rd nucleotide of tRNA anticodon (5’ end) may be capable of forming H bonds w/ more than 1 kind of 3rd nucleotide (3’ end) of codon

48. Reverse? • Howard Temin – proposed DNA provirus formed as intermediate in replication of RNA tumor viruses • RNA-directed DNA polymerase (Reverse transcriptase) – made DNA from RNA template • Retroviruses • HIV

49. Mutations • Changes in nucleotide sequence of DNA • Spontaneously during DNA replication, mitosis, meiosis, or because of mutagens • Low rate of occurrence because of cell’s repair mechanisms • Provide diversity of genes • Variation • evolution • Copied as normal, no greater chance of further mutation (normally)

50. Base substitution mutation • Simplest • 1 pair nucleotides changes • From errors in base pairing during replication • Affects transcribed mRNA and polypeptide