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Chapter 12: RNA and Protein Synthesis

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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 Nuclear envelope DNA TRANSCRIPTION Pre-mRNA mRNA TRANSLATION Ribosome
Fig. 17-3b-3 Nuclear envelope DNA TRANSCRIPTION Pre-mRNA RNA PROCESSING mRNA Figure 17.3 Overview: the roles of transcription and translation in the flow of genetic information TRANSLATION Ribosome Polypeptide (b) Eukaryotic cell

4 Gene 2 Gene 1 Gene 3 DNA template strand mRNA Codon TRANSLATION
Fig. 17-4 Gene 2 DNA molecule Gene 1 Gene 3 DNA template strand TRANSCRIPTION Figure 17.4 The triplet code 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 Several transcription factors must bind to the DNA before RNA
Fig. 17-8 1 A eukaryotic promoter includes a TATA box Promoter Template 5 3 3 5 TATA box Start point Template DNA strand 2 Several transcription factors must bind to the DNA before RNA polymerase II can do so. Transcription factors 5 3 3 5 3 Additional transcription factors bind to the DNA along with RNA polymerase II, forming the transcription initiation complex. Figure 17.8 The initiation of transcription at a eukaryotic promoter RNA polymerase II Transcription factors 5 3 3 5 5 RNA transcript Transcription initiation complex

11 Completed RNA transcript
Fig. 17-7a-4 Promoter Transcription unit 5 3 3 5 DNA Start point RNA polymerase 1 Initiation 5 3 3 5 RNA transcript Template strand of DNA Unwound DNA 2 Elongation Rewound DNA 5 3 3 3 5 Figure 17.7 The stages of transcription: initiation, elongation, and termination 5 RNA transcript 3 Termination 5 3 3 5 5 3 Completed RNA transcript

12 Nontemplate Elongation strand of DNA RNA nucleotides RNA polymerase 3
Fig. 17-7b Elongation Nontemplate strand of DNA RNA nucleotides RNA polymerase 3 3 end 5 Figure 17.7 The stages of transcription: initiation, elongation, and termination 5 Direction of transcription (“downstream”) Template strand of DNA Newly made RNA

13 Resulting mRNA Leader sequence – Coding 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 nucleotides long Enzymes add cap to 5’ end of mRNA Need cap for eukaryotic ribosome to bind May protect from degradation

16 Protein-coding segment Polyadenylation signal 5 3
Fig. 17-9 Protein-coding segment Polyadenylation signal 5 3 G P P P AAUAAA AAA … AAA 5 Cap 5 UTR Start codon Stop codon 3 UTR Poly-A tail Figure 17.9 RNA processing: addition of the 5 cap and poly-A tail

17 Polyadenylated (poly-A) tail gets added
3’ end When transcript complete, enzymes in nucleus recognize polyadenylation signal and cut mRNA at that site 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 exons spliced together Coding segment
Fig 5 Exon Intron Exon Intron Exon 3 Pre-mRNA 5 Cap Poly-A tail 1 30 31 104 105 146 Introns cut out and exons spliced together Coding segment mRNA 5 Cap Poly-A tail 1 146 Figure RNA processing: RNA splicing 5 UTR 3 UTR

21 RNA transcript (pre-mRNA) 5 Exon 1 Intron Exon 2
Fig RNA transcript (pre-mRNA) 5 Exon 1 Intron Exon 2 Protein Other proteins snRNA snRNPs Spliceosome 5 Figure The roles of snRNPs and spliceosomes in pre-mRNA splicing Spliceosome components Cut-out intron mRNA 5 Exon 1 Exon 2

22 mRNA processing

23 Amino acids tRNA with amino acid attached Ribosome tRNA Anticodon 5
Fig Amino acids Polypeptide tRNA with amino acid attached Ribosome Trp Phe Gly Figure Translation: the basic concept tRNA Anticodon 5 Codons 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 Aminoacyl-tRNA Amino acid synthetase (enzyme) tRNA Aminoacyl-tRNA
Fig 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 Figure An aminoacyl-tRNA synthetase joining a specific amino acid to a 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 (a) Two-dimensional structure
Fig a 3 Amino acid attachment site 5 Hydrogen bonds Figure The structure of transfer RNA (tRNA) Anticodon (a) Two-dimensional structure

30 Translation – At Ribosomes
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 (b) Schematic model showing binding sites
Fig b 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 Figure The anatomy of a functioning ribosome 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 Translation initiation complex
Fig Large ribosomal subunit 3 U C 5 A P site Met 5 A Met U G 3 Initiator tRNA GTP GDP E A mRNA 5 5 3 3 Start codon Figure The initiation of translation Small ribosomal subunit mRNA binding site Translation initiation complex

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 GDP GDP Amino end of polypeptide E 3 mRNA Ribosome ready for
Fig Amino end of polypeptide E 3 mRNA Ribosome ready for next aminoacyl tRNA P site A site 5 GTP GDP E E P A P A Figure The elongation cycle of translation 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 Release factor Free polypeptide 5 3 3 3 2 5 5 Stop codon
Fig Release factor Free polypeptide 5 3 3 3 2 5 5 GTP Stop codon (UAG, UAA, or UGA) 2 GDP Figure The termination of translation

41 Translation

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

43 Fig. 17-3 DNA TRANSCRIPTION mRNA Ribosome TRANSLATION Polypeptide (a) Bacterial cell Nuclear envelope DNA TRANSCRIPTION Pre-mRNA RNA PROCESSING Figure 17.3 Overview: the roles of transcription and translation in the flow of genetic information 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 First mRNA base (5 end of codon) Third mRNA base (3 end of codon)
Fig. 17-5 Second mRNA base First mRNA base (5 end of codon) Third mRNA base (3 end of codon) Figure 17.5 The dictionary of the genetic code

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

51 Missense mutations Base substitutions that result in the replacement of 1 AA by another Wide range of effects Enzyme activity “silent” – substituted w/ closely related AA, effects undetectable

52 Nonsense mutations Base substitution that converts an AA-specifying codon to a termination codon Usually destroys function of gene product

53 Frameshift mutations 1 or 2 nucleotide pairs are inserted or deleted from the molecule, causing an alteration of the reading frame Codons downstream now specify entirely new sequence of AA Different effects depends where it happens Entirely new polypeptide Stop codon w/in short distance of mutation Loss enzyme activity (disastrous)

54 Figure 17.23 Types of point mutations
Wild-type DNA template strand 3 5 5 3 mRNA 5 3 Protein Stop Amino end Carboxyl end A instead of G Extra A 3 5 3 5 5 3 5 3 U instead of C Extra U 5 3 5 3 Stop Stop Silent (no effect on amino acid sequence) Frameshift causing immediate nonsense (1 base-pair insertion) T instead of C missing 3 5 3 5 5 3 5 3 A instead of G missing 5 3 5 3 Stop Figure Types of point mutations Missense Frameshift causing extensive missense (1 base-pair deletion) A instead of T missing 3 5 3 5 5 3 5 3 U instead of A missing 5 3 5 3 Stop Stop Nonsense No frameshift, but one amino acid missing (3 base-pair deletion) (a) Base-pair substitution (b) Base-pair insertion or deletion

55 Jumping Genes (mobile genetic elements, transposable elements, transposons) DNA sequence “jumps” to middle of gene, disrupting gene functions and/or activation previously inactive genes Genes spontaneously turned on or off Barbara McClintock – 1950s Require transposase enzyme

56 Mutagens Mutagens Carcinogens Agents that cause mutations
Radiation – X, gamma, cosmic, UV rays; chemicals Carcinogens Cause cancer

57 Fig DNA TRANSCRIPTION 3 Poly-A RNA polymerase 5 RNA transcript RNA PROCESSING Exon RNA transcript (pre-mRNA) Intron Aminoacyl-tRNA synthetase Poly-A NUCLEUS Amino acid AMINO ACID ACTIVATION CYTOPLASM tRNA mRNA Growing polypeptide Cap 3 A Activated amino acid Poly-A P Ribosomal subunits Figure A summary of transcription and translation in a eukaryotic cell E Cap 5 TRANSLATION E A Anticodon Codon Ribosome

58 Fig. 17-UN8

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