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

• 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

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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

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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

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

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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

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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)

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

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

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

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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

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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

Crystal structure of T. thermophilus ribosome 30S shows rRNA domains

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Fig. 9 rRNA domains:H = head; N = neck; B = beak; Sh = shoulder;P = platform; Bo = body;Sp = spur

stereo

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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)

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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

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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

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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

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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

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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

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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

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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

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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

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Modified Nucleosides occur in tRNA

Fig. 25 Modifications occur during processing of tRNA; many enzymes required

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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

Charging tRNA with amino acid: requires ATP, aminoacyl tRNA synthase

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Fig. 17.2

Changing amino acid chemically after charging results in insertion of wrong amino acid

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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

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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

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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

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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

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’).

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