The Adaptor Hypothesis
Among Francis Crick's great contributions in the area of the Genetic Code and the expression of the genetic information were the enunciation of The Adaptor Hypothesis and, later on, The Wobble Hypothesis.
The Adaptor Hypothesis arose when Crick analyzed some ideas of George Gamow, generalized them and explained them in a paper he wrote for the (exclusive?) RNA Tie Club. The paper was never published in a "proper" journal, but Crick states in What Mad Pursuit that "it is my most influential unpublished paper":
"The main idea was that it was very difficult to consider how DNA or RNA, in any conceivable form, could provide a direct template for the side-chains of the twenty standard amino acids. What any structure was likely to have was a specific pattern of atomic groups that could form hydrogen bonds. I therefore proposed a theory in which there were twenty adaptors (one for each amino acid), together with twenty special enzymes. Each enzyme would join one particular amino acid to its own special adaptor. This combination would then diffuse to the RNA template. An adaptor molecule could fit in only those places on the nucleic acid template where it could form the necessary hydrogen bonds to hold it in place. Sitting there, it would have carried its amino acid to just the right place where it was needed."
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As we now know, tRNA functions as the adaptor molecules (work of Paul Zamecnik and Mahlon Hoagland) and aminoacyl-tRNA synthetases (AARS) are the very special enzymes that attach the correct amino acid to each tRNA. There are 21 aminoacyl-tRNA synthetases in E. coli (one for each amino acid except lysine, which has two). Early notions that led to the belief that there was one AARS for each of the twenty amino acids have now been discounted with the discovery that some organism with fewer than 20 AARS's modify amino acids after they have been added to tRNA.
How tRNAs can specify 61 codons is given by Crick's Wobble Hypothesis.
Transfer RNA
Transfer RNAs are small molecules ranging in size from 73 to 93 nucleotides in length. They all have a characteristic cloverleaf type of secondary structure:
Image from the MIT Biology Hypertextbook Chapter on The Central Dogma of Biology.
This structure was first recognized by Robert Holley (right - image from Nobel web site) in 1965. He determined the nucleotide sequence of the yeast tRNAAla which is 76 nt in length. He shared the 1968 Nobel Prize in Physiology or Medicine with Marshall Nirenberg and with Har Gobind Khorana .
The nucleotide sequences of many other tRNAs have since been determined. They share the following general features:
- A cloverleaf secondary structure which has the following parts:
The Amino Acid Arm or Acceptor Stem.
In this stem, the 5' and 3' ends of the tRNA are base-paired. The amino acid specific for the tRNA is covalently attached to a 3' OH group on the terminal adenine nucleotide.
The DHU Arm
This arm frequently contains the modified base dihydrouracil.
The Anticodon Arm
This arm contains the anticodon triplet exposed in a loop region.
An Extra Arm
As the name implies, this arm is not always present. It can be of variable length and is largely responsible for the variation noted in the length of tRNAs.
The T-Y-C Arm
This arm contains the conserved sequence of the three ribonucleotides, ribothymidine, pseudouridine and cytosine.
- At least 8 modified ribonucleotides.
In addition to those noted above, modified nucleotides are frequently found in tRNAs. An important one is Inosine which is often found as the 5' nucleotide of the anticodon.
- A 5' terminal guanine nucleotide, for the most part.
- A 3' terminal CCA trinucleotide.
- A conserved L-shaped tertiary structure.
The three dimensional structure of tRNAPhe was determined in 1974 by Alex Rich. The L-shape of the structure is formed through the interaction of bases in the DHU arm with those in the T-Y-C arm. These interactions include Watson-Crick base pairing, Hoogsteen base pairing, and triple-helical base pairing.
The following image, at the RNA Interest Group at the NIH in Washington, shows a detailed molecular view of a tRNA molecule:
tRNA processing
Many tRNA genes (in both eukaryotes and prokaryotes) are synthesized as part of larger precursor molecules. The mature final forms of these RNA molecules are obtained by processing reactions that involve both the removal of nucleotides and, in some instances, by the addition of nucleotides.
E. coli has 7 rRNA operons, each of which codes for a 16S, a 23S and a 5S rRNA as well as a variety of tRNAs:
OPERON tRNA GENES rrnA tRNA-Ile, tRNA-Ala rrnB tRNA-Glu rrnC tRNA-Glu, tRNA-Asp, tRNA-Trp rrnD tRNA-Ala, tRNA-Ile, tRNA-Thr rrnE tRNA-Glu rrnG tRNA-Glu rrnH tRNA-Ala, tRNA-Ile, tRNA-Asp E. coli also has a number of other operons that consist of or contain tRNA genes.
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tRNA precursor molecules are processed by a series of specific ribonucleases:
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- Ribonuclease D removes nucleotides from the 3' end to within two of the CCA sequence.
- Ribonuclease P then cleaves and generates the 5' end. This enzyme contains a protein of size 20 kD and a 377 nt RNA (M1 RNA) which contains the catalytic activity. Sidney Altman was awarded the 1989 Nobel Prize in Chemistry (shared with TomCech) for his demonstration that the M1 RNA has true catalytic activity.
- Ribonuclease D then removes the final two nucleotides from the 3' end of the tRNA.
Some tRNA genes do not code for the 3' terminal CCA. In these cases, the CCA is added by a special enzyme.
Some tRNA genes contain introns. Some of these are capable of self-splicing (e.g. those in chloroplasts and cyanobacteria). Others, such as those in yeast, have their own enzymatic mechanism for removal.
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The Wobble Hypothesis
In 1965, Robert Holley determined the sequence of yeast tRNAAla. The sequence of the anticodon bases in this tRNA surprised them. It contained the nucleotide Inosine at the 5' position in the anticodon.
Inosine (I) is the nucleotide of the base hypoxanthine, a deaminated adenine. Inosine monophosphate (IMP) is actually a precursor in the biosynthesis of purine nucleotides.
In 1966, Francis Crick proposed the Wobble hypothesis to generalize this observation. He suggested that while the interaction between the codon in the mRNA and the anticodon in the tRNA needed to be exact in two of the three nucleotide positions, this did not have to be so in the third position. He proposed that non-standard base-pairing might occur between the nucleotide base in the 5' position of the anticodon and the 3' position of the codon.
This hypothesis not only accounts for the number of tRNAs that are observed, it also accounts for the degeneracy that is observed in the Genetic Code. The degenerate base is that in the wobble position.
Crick recognised that the following base-pair schemes were possible:
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5' anticodon base 3' codon base A U C G G C or U U A or G I A or C or U These wobble rules are not followed exactly. If they were, only 31 tRNAs would be needed to pair will all possible codons. There are, however, more than 31 tRNAs.
The sequence of Escherichia coli K12 contains 84 tRNA reading frames. The only amino acids with a single tRNA are histidine, tryptophan and selenocysteine. There are 7 tRNAs for arginine and valine, and there are 8 tRNAs for leucine.
You can view a table of all non-protein encoding RNA species in E. coli at http://www.genetics.wisc.edu/html/orftables/rnatable.html Finally, although Holley determined the sequence of yeast tRNAAla in 1965, it was not until 1999 that the gene responsible for generating the inosine nucleotide at the wobble position was identified. André Gerber and Walter Keller from the Dept. of Cell Biology at the University of Basel demonstrated that an open reading frame (ORF) in the sequence of the yeast genome coded for the adenosine deaminase that generates inosine at the wobble position of tRNAs.
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