1. Chapt 19: Ribosomes and Transfer RNA
Student 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
tRNA (pink) and aa tRNA synthetase

2. 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

3. Bacterial Ribosome Composition
E. coli ribosome 70S
30 subunit:
16S rRNA
21 proteins (S1 – S21)
50S subunit:
5S rRNA
23S rRNA
34 proteins (L1 – L34)
Eukaryotic organelle ribosomes are similar, smaller
Fig. 3.16

4. 19.1 Bacterial Ribosomes 30S - 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

5. 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

6. Elongation: peptidyl transferase of 50S joins amino acids in peptide bond
GTP and many protein factors are required;
Incoming aa-tRNA receives growing polypeptide chain
Translocation and exit of empty tRNA
Fig. 3.19

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)

8. Fig. 19.1 Thermus thermophilus a-d rotated versions; 30S front in a
e, top with 50S top;
f, g individual 50S, 30S
16S rRNA cyan
23S rRNA gray
5S RNA dark blue
tRNA gold, orange

9. Fig. 2 tRNA bound to codons on ribosome
Fig. 3 structure of ribosome showing tRNAs bound at interface of subunits

10. Ribosomal proteins identified by 2D gel electrophoresis
More sensitive than 1D:
1st dimension pH 8.6, 8% acrylamide gel
2nd dimension, pH % acrylamide
Also cloned genes and purifed proteins
Fig. 5 E. coli proteins

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

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

13. Crystal structure of T. thermophilus ribosome 30S shows rRNA domains
stereo
Fig. 9 rRNA domains:
H = head; N = neck;
B = beak; Sh = shoulder;
P = platform; Bo = body;
Sp = spur

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)

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 S of Archaeon Haloarcula; green is peptidyl transferase region; yellow proteins

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

17. 50S Exit Tunnel Exit tunnel through 50S subunit
Fig. 20
Exit tunnel through 50S subunit
Just wide enough to allow protein a-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)

18. Polysomes mRNAs translated by > one ribosome at at time
Fig. 21 polysomes in bacteria; transcirption and translation simultaneously
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]

19. 19.2 Transfer RNA
Fig. 24 tRNA
molecule
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

20. Discovery of tRNA Small, independent of ribosome
3’ CCA-aa 5’
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)

21. tRNA Structure: cloverleaf
common secondary structure
4 base-paired stems define 3 stem-loops
D loop - dihydrouracil
Anticodon loop
T loop (TYC sequence – Y = pseudouridine)
Acceptor stem - site amino acids are added

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

23. Modified Nucleosides occur in tRNA
Fig. 25 Modifications occur during processing of tRNA; many enzymes required

24. 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
Fig. 17.1

25. Charging tRNA with amino acid: requires ATP, aminoacyl tRNA synthase
Fig. 17.2

26. Changing amino acid chemically after charging results in insertion of wrong amino acid
Ribosome recognizes tRNA not the amino acid
Fig. 28 chemically altered cys in cys-tRNA resulted in incorrect amino acid ala being inserted in synthetic mRNA

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

28. Structures of Synthetase-tRNA Complexes
Interactions differ between 2 classes of aminoacyl-tRNA synthetases: opposite sides
Class I approach D loop, minor groove of acceptor stem
E.g. GlnRS-tRNAgln
B) class 2 bind variable region, major groove of acceptor stem
E.g. AspRS-tRNAasp
Fig. 30

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

30. 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’).