Chapt 19: Ribosomes and Transfer RNA Student learning outcomes Describe basic structure of the...
-
Upload
theodore-casey -
Category
Documents
-
view
215 -
download
1
Transcript of Chapt 19: Ribosomes and Transfer RNA Student learning outcomes Describe basic structure of the...
![Page 1: Chapt 19: Ribosomes and Transfer RNA Student learning outcomes Describe basic structure of the ribosome, relationship of two subunits - catalytic roles.](https://reader036.fdocuments.us/reader036/viewer/2022062515/56649ce35503460f949af79c/html5/thumbnails/1.jpg)
Chapt 19: Ribosomes and Transfer RNAStudent learning outcomes• Describe basic structure of the ribosome,
relationship of two subunits - catalytic roles of RNA
• Describe basic structure of tRNA• Explain how amino acyl tRNA synthetases provide
second code – insert correct amino acid on tRNA
• Recall principles of translation,
aa joined in peptide bond
while bound in P and A sites • peptidyl transferase reaction
19-1tRNA (pink) and aa tRNA synthetase
![Page 2: Chapt 19: Ribosomes and Transfer RNA Student learning outcomes Describe basic structure of the ribosome, relationship of two subunits - catalytic roles.](https://reader036.fdocuments.us/reader036/viewer/2022062515/56649ce35503460f949af79c/html5/thumbnails/2.jpg)
• Appreciate Nobel Prizes for 2010 for ribosome structure and function:– Tom Steitz 50S ribosome structure Haloarcula– Venkatraman Ramakrishnan – 30S structure Thermus
thermophilus– Ada Yonath – 30S structure Thermus thermophilus,
started work crystallography Geobacillus, Haloarcula –
• Important Figures: 1, 2, 3*, 7, 8, 14, 15, 18, 20, 22, 24, 25, 26*, 28*, 31
• Review problems: 1, 3, 4, 5, 6, 8, 13, 14, 15, 19; AQ 1, 2
19-2
![Page 3: Chapt 19: Ribosomes and Transfer RNA Student learning outcomes Describe basic structure of the ribosome, relationship of two subunits - catalytic roles.](https://reader036.fdocuments.us/reader036/viewer/2022062515/56649ce35503460f949af79c/html5/thumbnails/3.jpg)
19-3Fig. 3.16
• E. coli ribosome 70S• 30 subunit:
– 16S rRNA– 21 proteins (S1 – S21)
• 50S subunit:– 5S rRNA– 23S rRNA– 34 proteins (L1 – L34)
Bacterial Ribosome Composition
Eukaryotic organelle ribosomes are similar, smaller
![Page 4: Chapt 19: Ribosomes and Transfer RNA Student learning outcomes Describe basic structure of the ribosome, relationship of two subunits - catalytic roles.](https://reader036.fdocuments.us/reader036/viewer/2022062515/56649ce35503460f949af79c/html5/thumbnails/4.jpg)
19-4
19.1 Bacterial Ribosomes30S - small subunit
decodes mRNA
50S –large subunit links amino acids together through peptide bonds
• Eukaryotic cytoplasmic ribosomes: – Larger (80S,- 40S, 60S– more RNAs, more proteins– 28S, 18S, 5.8S, 5S
Fig. 4 Ribosome with 3 tRNAs in A (aminoacyl), P (peptidyl) and E (exit) sites
![Page 5: Chapt 19: Ribosomes and Transfer RNA Student learning outcomes Describe basic structure of the ribosome, relationship of two subunits - catalytic roles.](https://reader036.fdocuments.us/reader036/viewer/2022062515/56649ce35503460f949af79c/html5/thumbnails/5.jpg)
Brief recall Protein synthesis• Prokaryotes: polycistronic
– mRNA binds 30S subunit at ribosome binding site– 1st tRNA is fmet (N-formyl-methionine) in P site– Lots of protein factors (IF, EF), GTP help– 50S subunit binds– 2nd tRNA binds to A site; peptide bond forms– Translocation of tRNA-peptide to E site
Eukaryotes: monocistronic– Ribosomes bind CAP, scan to find 1st AUG– 1st tRNA is met, not fmet
19-5
![Page 6: Chapt 19: Ribosomes and Transfer RNA Student learning outcomes Describe basic structure of the ribosome, relationship of two subunits - catalytic roles.](https://reader036.fdocuments.us/reader036/viewer/2022062515/56649ce35503460f949af79c/html5/thumbnails/6.jpg)
Elongation: peptidyl transferase of 50Sjoins amino acids in peptide bond
19-6Fig. 3.19
GTP and many protein factors are required;
Incoming aa-tRNA receives growing polypeptide chain
Translocation and exit of empty tRNA
![Page 7: Chapt 19: Ribosomes and Transfer RNA Student learning outcomes Describe basic structure of the ribosome, relationship of two subunits - catalytic roles.](https://reader036.fdocuments.us/reader036/viewer/2022062515/56649ce35503460f949af79c/html5/thumbnails/7.jpg)
19-7
Fine Structure of 70S Ribosome
• Bacterial Thermus thermophilus crystal structure: 70S ribosome with mRNA analog, 3 tRNAs :– Positions, tertiary structures of all 3 rRNAs, most proteins– Shapes and locations of tRNAs in A, P, and E sites– Binding sites for tRNAs in ribosome are rRNA, not protein– Contacts between subunits are mostly rRNA
– Anticodons of tRNAs in A and P sites approach each other closely enough to base-pair with adjacent codons bound to 30S subunit as mRNA kinks 45° (Fig. 2)
![Page 8: Chapt 19: Ribosomes and Transfer RNA Student learning outcomes Describe basic structure of the ribosome, relationship of two subunits - catalytic roles.](https://reader036.fdocuments.us/reader036/viewer/2022062515/56649ce35503460f949af79c/html5/thumbnails/8.jpg)
Fig. 19.1 Thermus thermophilusa-d rotated versions;
30S front in ae, top with 50S top;f, g individual 50S, 30S
16S rRNA cyan23S rRNA gray5S RNA dark bluetRNA gold, orange
![Page 9: Chapt 19: Ribosomes and Transfer RNA Student learning outcomes Describe basic structure of the ribosome, relationship of two subunits - catalytic roles.](https://reader036.fdocuments.us/reader036/viewer/2022062515/56649ce35503460f949af79c/html5/thumbnails/9.jpg)
19-9
Fig. 2 tRNA bound to codons on ribosome Fig. 3 structure of ribosome showing tRNAs bound at interface of subunits
![Page 10: Chapt 19: Ribosomes and Transfer RNA Student learning outcomes Describe basic structure of the ribosome, relationship of two subunits - catalytic roles.](https://reader036.fdocuments.us/reader036/viewer/2022062515/56649ce35503460f949af79c/html5/thumbnails/10.jpg)
Ribosomal proteins identified by 2D gel electrophoresis
• More sensitive than 1D:• 1st dimension pH 8.6, 8%
acrylamide gel• 2nd dimension, pH 4.6 18%
acrylamide
• Also cloned genes and purifed proteins
19-10Fig. 5 E. coli proteins
![Page 11: Chapt 19: Ribosomes and Transfer RNA Student learning outcomes Describe basic structure of the ribosome, relationship of two subunits - catalytic roles.](https://reader036.fdocuments.us/reader036/viewer/2022062515/56649ce35503460f949af79c/html5/thumbnails/11.jpg)
19-11
Ribosome Assembly
• E. coli assembly with purified proteins in vitro :
• 30S ribosomal subunit begins with 16S rRNA
• Proteins join sequentially and cooperatively– Proteins added early in
process help later proteins to bind to growing particle
Fig. 20, thick arrows strong facilitating, thin weaker
![Page 12: Chapt 19: Ribosomes and Transfer RNA Student learning outcomes Describe basic structure of the ribosome, relationship of two subunits - catalytic roles.](https://reader036.fdocuments.us/reader036/viewer/2022062515/56649ce35503460f949af79c/html5/thumbnails/12.jpg)
19-12
Fine Structure of 30S Subunit
• Consensus sequences of 16S rRNA led to secondary structure
• X-ray crystallography studies confirmed– 30S subunit - extensively
base-paired 16S rRNA shape essentially outlines particle
– X-ray crystallography confirmed locations of 30S ribosomal proteins
– Three major domains
Fig. 8 T. thermophilus 16S
![Page 13: Chapt 19: Ribosomes and Transfer RNA Student learning outcomes Describe basic structure of the ribosome, relationship of two subunits - catalytic roles.](https://reader036.fdocuments.us/reader036/viewer/2022062515/56649ce35503460f949af79c/html5/thumbnails/13.jpg)
Crystal structure of T. thermophilus ribosome 30S shows rRNA domains
19-13
Fig. 9 rRNA domains:H = head; N = neck; B = beak; Sh = shoulder;P = platform; Bo = body;Sp = spur
stereo
![Page 14: Chapt 19: Ribosomes and Transfer RNA Student learning outcomes Describe basic structure of the ribosome, relationship of two subunits - catalytic roles.](https://reader036.fdocuments.us/reader036/viewer/2022062515/56649ce35503460f949af79c/html5/thumbnails/14.jpg)
19-14
30S Subunit binds antibiotics, initiation factors
• 2 roles of 30S ribosomal subunit:– Facilitates proper decoding between codons and
aminoacyl-tRNA anticodons– Also participates in translocation
• Crystal structures of 30S subunits with interfering antibiotics sheds light on translocation and decoding– Spectinomycin – interferes with translocation– Streptomycin – error rate increases– Paromomycin – decreases accuracy of translation (A site)
Antibiotic-resistant mutants can arise from altered ribosomal proteins (S12)
30S binds initiation factors (IF)
![Page 15: Chapt 19: Ribosomes and Transfer RNA Student learning outcomes Describe basic structure of the ribosome, relationship of two subunits - catalytic roles.](https://reader036.fdocuments.us/reader036/viewer/2022062515/56649ce35503460f949af79c/html5/thumbnails/15.jpg)
19-15
Fine Structure of 50S Subunit - Steitz
• Crystal structure to 2.4 Å• Relatively few proteins at
interface between ribosomal subunits– No proteins within 18 Å of
peptidyl transferase active center (tagged with transition state analog)
– 2’-OH group of tRNA in P site forms H bond to amino group of aminoacyl-tRNA in A site
Fig.16 50S of Archaeon Haloarcula; green is peptidyl transferase region; yellow proteins
![Page 16: Chapt 19: Ribosomes and Transfer RNA Student learning outcomes Describe basic structure of the ribosome, relationship of two subunits - catalytic roles.](https://reader036.fdocuments.us/reader036/viewer/2022062515/56649ce35503460f949af79c/html5/thumbnails/16.jpg)
19-16
Role of 2‘-OH of tRNA• 2’-OH group of tRNA in P site:
Forms H-bond to amino group
of aminoacyl-tRNA in A site
Helps catalyze peptidyl transferase reaction
• Removal of 2’-OH group eliminates peptidyl transferase activity (Fig. 19)
Fig. 18 Peptide bond involves Nucleophilic attack by aa in A site to COO- joined to tRNA in P site;
Amino acid joined to 3’-OH of tRNA
![Page 17: Chapt 19: Ribosomes and Transfer RNA Student learning outcomes Describe basic structure of the ribosome, relationship of two subunits - catalytic roles.](https://reader036.fdocuments.us/reader036/viewer/2022062515/56649ce35503460f949af79c/html5/thumbnails/17.jpg)
19-17
50S Exit Tunnel
Exit tunnel through 50S subunit– Just wide enough to allow protein -helix to pass– Walls of tunnel made of RNA– Hydrophilicity likely to allow exposed hydrophobic side
chains of nascent polypeptide to slide easily (not bind)
Fig. 20
![Page 18: Chapt 19: Ribosomes and Transfer RNA Student learning outcomes Describe basic structure of the ribosome, relationship of two subunits - catalytic roles.](https://reader036.fdocuments.us/reader036/viewer/2022062515/56649ce35503460f949af79c/html5/thumbnails/18.jpg)
19-18
Polysomes
• mRNAs translated by > one ribosome at at time• Polysome: structure in which many ribosomes
translate mRNA in tandem• Eukaryotic polysomes are found in cytoplasm• In Prokaryotes, transcription of gene and translation
of resulting mRNA occur simultaneously• [Many polysomes associated with active gene]
Fig. 21 polysomes in bacteria; transcirption and translation simultaneously
![Page 19: Chapt 19: Ribosomes and Transfer RNA Student learning outcomes Describe basic structure of the ribosome, relationship of two subunits - catalytic roles.](https://reader036.fdocuments.us/reader036/viewer/2022062515/56649ce35503460f949af79c/html5/thumbnails/19.jpg)
19-19
19.2 Transfer RNA
• Adaptor molecule (proposed by Crick, 1958) as mediator between string of nucleotides in DNA or RNA and string of amino acids in protein
• 3 nucleotides could pair with nucleotides in codons
Fig. 24 tRNAmolecule
![Page 20: Chapt 19: Ribosomes and Transfer RNA Student learning outcomes Describe basic structure of the ribosome, relationship of two subunits - catalytic roles.](https://reader036.fdocuments.us/reader036/viewer/2022062515/56649ce35503460f949af79c/html5/thumbnails/20.jpg)
19-20
Discovery of tRNA
• Small, independent of ribosome• Could be charged with amino
acid: covalently joined in process requiring ATP
• Charged species transfers amino acid to growing polypeptide:– Amino end of 2nd amino acid
attacks COO- of first aa (which COO- is joined through tRNA)
5’3’ CCA-aa
![Page 21: Chapt 19: Ribosomes and Transfer RNA Student learning outcomes Describe basic structure of the ribosome, relationship of two subunits - catalytic roles.](https://reader036.fdocuments.us/reader036/viewer/2022062515/56649ce35503460f949af79c/html5/thumbnails/21.jpg)
19-21
tRNA Structure:cloverleaf
• common secondary structure• 4 base-paired stems define 3 stem-loops
– D loop - dihydrouracil– Anticodon loop– T loop (TC sequence – = pseudouridine)
• Acceptor stem - site amino acids are added
![Page 22: Chapt 19: Ribosomes and Transfer RNA Student learning outcomes Describe basic structure of the ribosome, relationship of two subunits - catalytic roles.](https://reader036.fdocuments.us/reader036/viewer/2022062515/56649ce35503460f949af79c/html5/thumbnails/22.jpg)
19-22
tRNA Shape
• Common 3-D shape resembling inverted L• Maximizes stability by lining up base pairs:
– D stem to anticodon stem– T stem to acceptor stem
• Anticodon protrudes from side of loop– Anticodon shape base-pairs with mRNA codon
![Page 23: Chapt 19: Ribosomes and Transfer RNA Student learning outcomes Describe basic structure of the ribosome, relationship of two subunits - catalytic roles.](https://reader036.fdocuments.us/reader036/viewer/2022062515/56649ce35503460f949af79c/html5/thumbnails/23.jpg)
19-23
Modified Nucleosides occur in tRNA
Fig. 25 Modifications occur during processing of tRNA; many enzymes required
![Page 24: Chapt 19: Ribosomes and Transfer RNA Student learning outcomes Describe basic structure of the ribosome, relationship of two subunits - catalytic roles.](https://reader036.fdocuments.us/reader036/viewer/2022062515/56649ce35503460f949af79c/html5/thumbnails/24.jpg)
19-24
Fig. 17.1
Amino acyl tRNA synthases add amino acids – second genetic code
Structure of tRNA-amino acid
• Amino acid covalently joined to specific tRNA at terminal 3’-CCA sequence
• Amino terminal end of aa-2nd tRNA attacks COO- of 1st aa-tRNA to form peptide bond
![Page 25: Chapt 19: Ribosomes and Transfer RNA Student learning outcomes Describe basic structure of the ribosome, relationship of two subunits - catalytic roles.](https://reader036.fdocuments.us/reader036/viewer/2022062515/56649ce35503460f949af79c/html5/thumbnails/25.jpg)
Charging tRNA with amino acid: requires ATP, aminoacyl tRNA synthase
19-25
Fig. 17.2
![Page 26: Chapt 19: Ribosomes and Transfer RNA Student learning outcomes Describe basic structure of the ribosome, relationship of two subunits - catalytic roles.](https://reader036.fdocuments.us/reader036/viewer/2022062515/56649ce35503460f949af79c/html5/thumbnails/26.jpg)
Changing amino acid chemically after charging results in insertion of wrong amino acid
19-26
Fig. 28 chemically altered cys in cys-tRNA resulted in incorrect amino acid ala being inserted in synthetic mRNA
Ribosome recognizes tRNA not the amino acid
![Page 27: Chapt 19: Ribosomes and Transfer RNA Student learning outcomes Describe basic structure of the ribosome, relationship of two subunits - catalytic roles.](https://reader036.fdocuments.us/reader036/viewer/2022062515/56649ce35503460f949af79c/html5/thumbnails/27.jpg)
19-27
tRNA Acceptor Stem and anticodon are important for aa tRNA synthase
• Biochemical and genetic experiments demonstrated:
• acceptor stem recognized in tRNA by cognate aa-tRNA synthetase
• Changing one base pair in acceptor stem can change charging specificity– Second genetic code: charging correct amino acid
• Anticodon, is also important element in charging specificity
![Page 28: Chapt 19: Ribosomes and Transfer RNA Student learning outcomes Describe basic structure of the ribosome, relationship of two subunits - catalytic roles.](https://reader036.fdocuments.us/reader036/viewer/2022062515/56649ce35503460f949af79c/html5/thumbnails/28.jpg)
19-28
Structures of Synthetase-tRNA Complexes
Interactions differ between 2 classes of aminoacyl-tRNA synthetases: opposite sides
a)Class I approach D loop, minor groove of acceptor stem
a) E.g. GlnRS-tRNAgln
b)B) class 2 bind variable region, major groove of acceptor stem
a) E.g. AspRS-tRNAasp
Fig. 30
![Page 29: Chapt 19: Ribosomes and Transfer RNA Student learning outcomes Describe basic structure of the ribosome, relationship of two subunits - catalytic roles.](https://reader036.fdocuments.us/reader036/viewer/2022062515/56649ce35503460f949af79c/html5/thumbnails/29.jpg)
19-29
aa- tRNA synthetases also proofread and edit
aa selectivity controlled by double-sieve mechanism– 1st sieve is coarse, excluding amino acids too big
• Active site for activation of amino acids is just big enough for cognate amino acid, not larger amino acids
–2nd sieve is fine, degrades too small aminoacyl-AMPs • Editing site admits small aminoacyl-AMPs and hydrolyzes• Cognate aminoacyl-AMP is too big to fit editing site• Enzyme transfers activated amino acid to cognate tRNA
![Page 30: Chapt 19: Ribosomes and Transfer RNA Student learning outcomes Describe basic structure of the ribosome, relationship of two subunits - catalytic roles.](https://reader036.fdocuments.us/reader036/viewer/2022062515/56649ce35503460f949af79c/html5/thumbnails/30.jpg)
Review questions
3. What parts of tRNA interact with 30S? With 50S?
4,5. Why is it important that the anticodons in A & P sites, and that tRNA acceptor stems in A & P sites approach each other closely?
14. Draw cloverleaf tRNA structure and draw important structural elements.
Draw the charged tRNA with an amino acid; diagram how one aa-tRNA is joined to the growing peptide chain (remember 5’, 3’).
19-30