1. Molecular Biology Fourth Edition
Lecture PowerPoint to accompany
Molecular Biology Fourth Edition
Robert F. Weaver
Chapter 19
Ribosomes and Transfer RNA
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

2. 19.1 Ribosomes
E. coli ribosome is a two-part structure with a sedimentation coefficient of 70S
Two subunits of this structure:
30S is the small subunit that decodes mRNA (Chapter. 17)
50S subunit links amino acids together through peptide bonds (Chapter 18)

3. Eukaryotic Ribosomes Eukaryotic cytoplasmic ribosomes are:
Larger
More complex
Eukaryotic organellar ribosomes are smaller than prokaryotic ones

4. Ribosome Composition The E. coli 30 subunit contains
16S rRNA (SD sequence recognition)
21 proteins (S1 – S21)
E. coli 50S subunit contains
5S rRNA
23S rRNA (peptidyl transferase)
34 proteins (L1 – L34)
Eukaryotic cytoplasmic ribosomes are:
Larger
Contain more RNAs and proteins

5. Ribosome Assembly
Assembly of the 30S ribosomal subunit in vitro begins with 16S rRNA
Proteins join sequentially and cooperatively
Proteins added early in the process
Help later proteins to bind to the growing particle

6. Interaction of the 30S Subunit with Antibiotics
30S ribosomal subunit plays 2 roles
Facilitates proper decoding between codons and aminoacyl-tRNA anticodons
Also participates in translocation
Crystal structures of 30S subunits with 3 antibiotics interferring with these 2 roles shed light on translocation and decoding (codon-anticodon interaction)
Spectinomycin- inhibit translocation
Streptomycin- cause errors in translation
Paromomycin – increase error rate by another mechanism

7. Spectinomycin Spectinomycin binds to 30S subunit near the neck
At this site, binding interferes with movement of the head
Head movement is required for translocation

8. Streptomycin Streptomycin binds near the A site of 30S subunit
Binding stabilizes the ram (ribosome ambiguity) state of the ribosomes
Fidelity of translation is reduced:
Allowing noncognate aminoacyl-tRNAs to bind easily to the A site
Preventing the shift to the restrictive state that is necessary for proofreading

9. Paromomycin
Paromomycin binds in the minor groove of 16S rRNA H44 helix near the A site
This binding flips out bases A1492 and A1493 to stabilize base pairing between codon and anti-codon
Flipping out process normally requires energy
Paromomycin forces it to occur and keeps the stabilizing bases in place
State of the A site stabilizes codon-anticodon interaction, including interaction between noncognate codons and anticodons with decline in fidelity

10. Polysomes
Most mRNAs are translated by more than one ribosome at at time
A structure in which many ribosomes translate mRNA in tandem is called a polysome
Eukaryotic polysomes are found in the cytoplasm
In prokaryotes, transcription of a gene and translation of the resulting mRNA occur simultaneously
Many polysomes are found associated with an active gene

11. Fig

12. 19.2 Transfer RNA
An adaptor molecule was proposed that could serve as a mediator between string of nucleotides in DNA or RNA and the string of amino acids in the corresponding protein
The adaptor contained 2 or 3 nucleotides that could pair with nucleotides in codons

13. The Discovery of tRNA
Transfer RNA was discovered as a small species independent of ribosomes
Zamecnik and et al.,1957
pH5 enzyme fraction works with ribosomes to direct translation of added mRNAs.
This small species could be charged with an amino acid
That species could then pass the amino acid to a growing polypeptide

14. Charging of tRNA with an amino acid
Mix RNA with pH5 enzyme,
ATP and [14C]Leucine
Charging of tRNA with an amino acid

15. Mix [14C]Leucine-charged pH 5
RNA with microsome
Incorporation of leucine from leucyl-tRNA in to the
nascent protein on ribosome

16. tRNA Structure
All tRNAs share a common secondary structure represented by a cloverleaf
Four base-paired stems define three stem-loops
D loop
Anticodon loop
T loop
The acceptor stem is the site to which amino acids are added in the charging step

17. Fig

18. tRNA Shape
tRNAs share a common three-dimensional shape resembling an inverted L
This shape maximizes stability by lining up the base pairs:
In the D stem with those in the anticodon stem
In the T stem with those in the acceptor stem
Anticodon of the tRNA protrudes from the side of the anticodon loop
Anticodon is twisted into a shape that base-pairs with corresponding codon in mRNA

19. Modified Nucleosides in tRNA

20. Are tRNAs made with modified bases, or are the bases modified after transcription?
tRNA are made in the same way that other RNAs are made, with the four standard bases.
Multiple enzymes modify the bases

21. What effects, if any, do these modifications have on tRNA function?
At least two tRNAs have been made in vitro with four normal, unmodified bases, and they were unable to bind amino acids
Each modification may have subtle effects on the efficiency of charging and tRNA usage

22. Recognition of tRNA Acceptor Stem
Biochemical and genetic experiments have demonstrated the importance of the acceptor stem in recognition of a tRNA by its cognate aminoacyl-tRNA synthetase
Changing one base pair in the acceptor stem can change the charging specificity

23. AMP/amino acid
coupling
AMP/tRNA displacement

24. Ribosome recognize the tRNA but not the amino acid
Lipmann, Benzer, von Ehrenstein and et al, 1962
Cysteinyl-tRNAcys →Alanyl-tRNAcys
In vitro translation using poly(UGU) as template—this does not contain any codon for alanine
Codon for ala is GCN, cysteine is UGU
Alanine is incorporated instead of cysteine
Fig

25. It is the nature of the tRNA that matters
Fig
It is the nature of the tRNA that matters

26. The acceptor stem ? The anticodon ?
Given that the secondary and tertiary structures of all tRNA are essentially the same, what base sequences in tRNA do the synthetases recognize when they are selecting one tRNA out of a pool of over 20?
The acceptor stem ?
The anticodon ?

27. The acceptor stem
1973, Smith and Celis found: GlnRS and TyrRS binding to tRNA differ only in one base 73 from G to A in suppressor tRNA assay

28. The acceptor stem 1988, Hou and Schimmel found:
tRNAAla’s anticodn mutated to 5’-CUA-3’, so it inserts alanine in response to the amber codon UAG.
Insert amber codon in codon 10 of trpA gene
The mutation could be suppressed only by a tRNA that could insert an alanine in response to amber codon
Any other amino acid in position 10 yielded an inactive protein
G3-U70 base pair is a key determinant of
charging synthetases

29. 3

30. Further evidence tRNAcys/CUA and tRNAphe/CUA (CUA is anti-codon)
Has C3-G70 base pair in their acceptor stem
Change C3-G70 to G3-U70, then convert the tRNA to tRNAAla/CUA

31. The Anticodon
Biochemical and genetic experiments have shown that anticodon, like acceptor stem, is an important element in charging specificity
Sometimes the anticodon can be the absolute determinant of specificity

33. The first base in the anticodon (the wobble position) was the
Tab. 19.1
One different base
The first base in the anticodon (the wobble position) was the
most sensitive and had drastic effect on charging

34. Recognition of tRNAs by aminoacyl-tRNA synthetase:
The second genetic code

35. 1958, Pauling used thermodynamics and found:
Ile and val only differ in –CH2 group and one-fifth Val-tRNAile would be made
In fact, 1/150 amino acid is activated by IleRS to make Val and 1/3,000 aminoacyl-tRNA is Val-tRNAile

36. Structures of Synthetase-tRNA Complexes
Crystallography has shown that synthetase-tRNA interactions differ between the 2 classes of aminoacyl-tRNA synthetases
Class I synthetases
Pockets for acceptor stem and anticodon of their cognate tRNA
Approach the tRNAs from the D loop and acceptor stem minor groove side
Class II synthetases
Also have pockets for acceptor stem and anticodon
Approach tRNA from opposite including the variable arm and the major groove of the acceptor stem

37. Proofreading and Editing
Amino acid selectivity of at least some aminoacyl-tRNA synthetases is controlled by a double-sieve mechanism
1st sieve is coarse excluding amino acids too big
Enzyme accomplishes this with an active site for activation of amino acids just big enough to accommodate the cognate amino acid, not larger amino acids
2nd sieve is fine, degrades too small aminoacyl-AMPs
Done with a second active site, the editing site, admits small aminoacyl-AMPs and hydrolyzes them
Cognate aminoacyl-AMP is too big to fit into the editing site
Enzyme transfers the activated amino acid to its cognate tRNA

38. Fig

39. The space between Trp232 and Tyr386 is just big enough to
Fig
The space between Trp232 and Tyr386 is just big enough to
accommodate valine but not Ile (too big)
Abolish this region could abolish editing but not activation activity