Biochemistry I Fall Term, 2000

Lecture 21: Protein Synthesis: tRNA Structure & Charging

Assigned reading in Campbell: Chapter 9.1-9.3

Take the Review Quiz on Lecture 21 concepts.

Features of tRNA^Phe Structure is a page of JPEG images to accompany this lecture.

1. The Genetic Code
  A. Mutagenesis

1. Types of mutations

   a) transitions, i.e. R --> R' and Y --> Y'.   R = purine; Y = pyrimidine.

   b) transversions, i.e. R --> Y and vice versa.

2. Chemical mutagenesis

   a) 2-aminopurine causes transitions by substituting for A (or G).

   b) nitrous acid (HNO₂) causes transitions by deamination.
   Cytosine is converted to uracil: potential transition.
   Adenine is converted to hypoxanthine: potential transition.
   5-methylcytosine is converted to thymine.

3. Spontaneous mutagenesis

   a) deamination of C and 5-methylC causes transitions.

   b) the deamination rate increases with increasing temperature.

1. Codons are triplets: 4 different bases yields 43 = 64 different codons.
2. mRNA is decoded from 5' --> 3' producing protein N-term --> C-term.
3. tRNA's have anticodon segments that are complementary to the codons.
4. "UUU specifies Phe" was the first to be discovered.

   a) reconstituted protein synthesizing system. Nirenberg, et al.

   b) chemically synthesized RNA molecules: Khorana, et al.

   c) "AAA specifies Lys": a short story.

5. Punctuation

   a) AUG and GUG are start codons.

   b) UAG, UAA, and UGA are stop codons.

6. The code is highly degenerate, especially at the third position.
7. The code is (nearly) universal ... except for mitochondria.

2. tRNA: Structure and Aminoacylation
  A. Primary and Secondary Structures of tRNA.

1. Cloverleaf diagram of secondary structure

   a) Regions:

   Acceptor stem: amino acids are attached to the 3' terminus

   D arm: named for the dihydrouridine in the loop.

   Anticodon arm: contains the anticodon triplet.

   Variable loop: length is 3-21 nucleotides among tRNAs.

   TYC arm: named for the pseudouridine (Y) in the loop.

   b) Comparative sequence analysis. The invariant and semi-invariant positions correlate with important structural and functional features of tRNA. For example, base pairing and other base-base interactions were deduced before the structure was known.

2. Dozens of base (and sugar) modifications have been found.

1. Acceptor stem stacks on the TYC arm.
   Anticodon arm stacks on the D arm.
   The resulting coaxial helices are nearly normal A-form RNA.
2. Most of the bases (71/76) are stacked; only about half are base paired.
3. Mg²⁺ bound in the "corner of the L" stabilizes the structure.
4. Examples of non-standard base-base interactions.

   a) The "wobble" base pair at G4-U69.

   b) Base triple interactions; a base inserts into the deep groove of a standard base pair (distant in sequence).

   c) Intercalation of bases at two locations.

   These, and several other tertiary structure motifs, have also been found in the compact, folded structures of many other RNAs.

1. Amino acids are activated in the first step (Campbell, Fig. 9.5).
   
   This is usually an enzyme-bound intermediate.
2. Amino acids are attached to tRNA in the second step.
   
   The aminoacyl moiety is attached to the 3' OH (or 2' OH) of the -CCA end of the tRNA.

3. The overall energetics of tRNA charging is only slightly favorable. The aminoacyl-tRNA bond is in the "high energy" category. The overall reaction is driven by PP_i hydrolysis.

4. Fidelity of charging by editing or proofreading mechanisms.

   a) Editing in general:
   

   
     where EI is the activated intermediate (aminoacyl-AMP).
   

   b) Requirement for editing
     1) Consider IleRS mischarging of valine to Val-tRNA^Ile.
   Isoleucine binding in the first step is favored by only ~100-fold over valine. However, addition of tRNA^Ile leads to 100% hydrolysis of the incorrectly activated valyl-AMP intermediate.
     2) Hydrolytic editing occurs at a second active site. The "double sieve" sorts correct and incorrect charging. The first sieve invokes size and steric requirements. The second sieve invokes chemical characteristics.
   The overall fidelity is: (1 error/100 charging reactions)*(1 residual error/100 editing reactions) = 1*10^-4.

   c) Not all of the synthetases use editing steps, e.g. Tyr is sufficiently different from the other 19 amino acids (size and chemical characteristics), that substrate specificity alone ensures accurate charging.

1. Charged tRNAs are selected by ribosomes solely through codon-anticodon interactions.
2. Degeneracy, third position codon-anticodon pairing, and "wobble".

   Note the pairing combinations for tRNA^Phe:

   a) The anticodon is GAA.

   b) The complementary codons are UUC and UUU.

   c) The codon-anticodon pairing is antiparallel:
   
   

3. The anticodon nucleotides form a "mini half-helix" structure. Thus, the conformation is prepared for binding to codon nucleotides in mRNA on the ribosome.

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