Humboldt State University ® Department of Chemistry

Richard A. Paselk

Chem 432	Biochemistry	Spring 2002
Lecture Notes:: 10 April	© R. Paselk 2002
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Translation

tRNA

tRNA functions as an adaptor to correlate the four base nucleic acid language to the 20 amino acid language. One end of the folded molecule binds to the three-base codon on the messenger RNA while the other end is bound to an amino acid residue.

tRNA exhibits a hierarchical structure analogous to the structure of proteins:

• 1° = sequence of bases.
• 2° = double helical, H-bonded regions.
• 3° = overall 3-D folding.

Primary Structure: The sequence of tRNA is characterized by a number of conserved position when corrected for insertions:

• There are 15 invariant positions in the sequence occurring mostly in loops.
• In addition there are 8 semi-invariant positions which are fixed as either purines or pyrimidines occurring mostly in loops.

A characteristic of tRNAs is the high proportion of modified bases, up to 20%. These are all post-transcriptional modifications.

• Over 50 different modified bases have been found.
• So called "hypermodified" bases are usually found adjacent to the anticodon 3' nucleotide when it is A or U. These bases probably function by strengthening the base-pairing in the codon-anticodon via creation of a more non-polar environment.

The function of the modified bases is largely unknown. For example, tRNAs can function without modified bases, but bacteria lacking modified bases are less competitive than wild-types.

Secondary Structure: Almost all tRNAs can be arranged into a "clover leaf" structure which maximizes the potential H-bonding. This secondary structure has the following features, as exemplified by the structure of yeast phe-tRNA, Figure 26-6, p 852, in your text. [overhead]:

• The acceptor stem or amino acid stem. A seven base-pair 'stem' including the 5' and 3' ends which frequently includes non-Watson-Crick base-pairs.
  • The 5' end has a terminal phosphate.
  • The 3' end terminates in a CCA sequence with a 3'-OH.
    • The CCA sequence may be encoded by the DNA, or may be added post transcriptionally, depending on the specific tRNA.
• The D arm. A three or four base-pair stem ending in the D loop, so called because of the frequent presence of dihydrouridine (D) in the loop.
• The Anticodon arm. A five base-pair stem ending in the anticodon loop. This loop contains the three base anticodon which base-pairs with the codon on mRNA.
• The TYC arm. A five base-pair stem ending in the TYC loop, named because of the presence of an invariant TYC (Y=pseudouridine) sequence.
• The variable arm. This arm ranges from 3-21 bases and may include a stem with up to seven base-pairs.

Tertiary Structure: The tRNA secondary structure folds up into an "L-shaped" conformation with the acceptor site and codon loop at the ends of the two legs.

• The acceptor leg is composed of the acceptor stem and the TYC stem.
• The anticodon leg is composed of the D stem and the anticodon stem.
• Each leg is about 60Å (6 nm) long, with the acceptor and anticodon ends about 76Å apart.
• The structure is maintained by H-bonding and stacking interactions. The H-bonds include :
  • non-Watson-Crick base pairs,
  • ribose-OH H-bonds to other groups,
  • phosphate-O H-bonds to other groups.
• The bases in the resultant structure are largely inaccessible to water.

Aminoacyl tRNA Synthetases

Amino acid residues are covalently linked to tRNA in an "activated" form in a two reaction process:

1. Activation of the amino acid residue:
   
2. Formation of the aminoacyl-tRNA:
   

Note that the first reaction should have a free energy of about zero since we are breaking and forming acid anhydride bonds, an thus the reaction is driven by the subsequent hydrolysis of the pyrophosphate.

The second reaction is then driven to completion because the "activated" amino acid acid anhydride bond is broken and replaced with the relatively low energy ester bond.

Remarkably aminoacyl tRNA synthetases do not appear to be closely related to one-another (they have different sequences, and different folds!) - apparently they are so ancient they started independently. They exhibit a variety of quaternary structural patterns: a, a₂, a₄, and a₂b₂, with between 334 - >1000 amino acid residues. As another indicator of the great age of these proteins, the aminoacyl tRNA synthetases for the same amino acids are similar in evolutionarily diverse organisms, but the aminoacyl tRNA synthetases for different amino acids in the same organisms are generally dissimilar.

In the case of tyrosyl-tRNA synthetase the catalysis appears to operate strictly via transition state and proximity/orientation catalysis - there is no classical chemical catalysis (acid/base, covalent, etc.) apparent.

Most aminoacyl tRNA synthetase-tRNA contact sites are on the inner face of the 'L', but otherwise show no regularity. Some seem to recognize only the acceptor region, others the anticodon, etc. (see figures 26-10 to 26-13 in your text).

Finally, aminoacyl tRNA synthetases exhibit remarkable specificity by the use of editing in addition to substrate binding. For example, for isoleucyl tRNA synthetase:

• expect a differentiation between ilu and val of about 100 fold due to binding,
• in actuality see about a 50,000 fold discrimination in favor of ilu.
• Explanation is twofold for general case:
  • For amino acids larger than ilu, the size will prevent them from entering the active site, so high discrimination.
  • For amino acids like valine which are smaller, they can enter the active site and be attached to the tRNA, however a second, hydrolytic site is present on the enzyme which will allow the valyl residue to enter, but not isolucyl, thus valyl-tRNA is broken down, but isoleucyl-tRNA is released intact.

Wobble and Code Degeneracy

Even though there are isoaccepting tRNAs (different tRNAs specific for the same amino acid), it turns out that many tRNAs bind to a number of different codons specifying the same amino acid!

This observation is explained by the "wobble hypothesis" of Francis Crick. According to this model,

• the first two codon-anticodon pairings follow standard Watson-Crick rules (i.e AT and GC etc.),
• however, the third base pair can exhibit a certain amount of play or wobble, enabling a varieity of specific non-Watson-Crick pairings.

The various pairing possibilities allowed by wobble are shown in the table below:

Wobble Pairings (third anticodon/codon positions) 5'-anticodon base 3'-codon base C G A U U A or G G U or C I U, C, or A

• No cytosolic tRNAs are known which participate in non-wobble pairings.
• No cytosolic tRNAs are known with A in the 3rd anticodon position. Apparently the U-A pair is not permitted.

The wobble hypothesis requires at least 31 tRNAs to translate all 61 coding triplets plus one for special initiation tRNA. Most cells have >32. All isoaccepting tRNAs in a cell have the same amionoacyl tRNA synthetase.

Note that the most frequently used codons (those specifying the most frequently used amino acids) are complementary to the most abundant tRNA species.

Notes:

• Some mitochondrial tRNAs have more permissive wobble pairings. Some also have unusual structures.
• Selenocysteine is specified by UGA, apparently with the aid of context (local sequence) effects.
• Read about non-sense suppression (substitute an amino acid for a stop). Due to tRNA mutation.
  • Generally mutated tRNAs are minor members of isoaccepting sets, and protein stop factors compete, so most proteins are still terminated.

Ribosomes

Ribosomes are the machinery for protein translation. As noted in the table [overhead] (Tables 26-4, p 862 and 26-5, p 866) the ribosome is a very large and complex suprmolecular structure, approximately 2/3 RNA and 1/3 protein. Note that though quite similar overall, the eukaryotic particles are significantly larger, with larger main RNAs and an additional 5.8 s RNA and over 50% more proteins than the baterial ribosome.

Note also that the ribosomes are self-assembling particles.

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Last modified 22 April 2002