MCB 201 Gene Expression - Spring Semester 2004
Lecture 6 (The Three Roles of RNA in Translation continued)
1. Figure 4-22, Lodish4e: Assigning codons using synthetic mRNAs containing a single ribonucleotide. This is the strategy employed in the famous Nirenberg and Matthei experiments in the early 1960's in which a synthetic RNA polymer composed of only one type of nucleotide was used to program bacterial extracts that contained all the components needed for protein synthesis except mRNA. The result was the synthesis of polypeptides containing only a single type of amino acid. Additional refinements of this experimental strategy in which synthetic RNA with different combinations of nucleotides were used allowed these investigators to conclude that the code was read in groups of three nucleotides and to identify new codons.
2. Figure 4-24, Lodish4e: Solving the entire genetic code using chemically synthesized trinucleotides representing all possible codons. See Classic Experiment "Cracking the Genetic Code" at the textbook website. In this experiment, 20 ribosome-free bacterial extracts were prepared, each containing all possible amino acyl-tRNAs (aa-tRNA), but in each extract only one type of tRNA was charged with a radioactive amino acid, and this was a different one for each of the 20 extracts. The experiment is based on the fact that these aa-tRNAs and trinucleotide codons will pass through a nitrocellulose filter. However, ribosomes are retained on the filter, so in the presence of the appropriate trinucleotide codon, a stable complex of ribosome, codon and radioactive aa-tRNA (which will interact with the trinucleotide codon using its anticodon sequence) will be formed and captured on the filter. The filters can then be counted in a liquid scintillation counter to measure the amount of bound radioactivity.
3. Figure 4-21, Lodish5e: Translation of nucleic acid sequences in mRNA into amino acid sequences in proteins requires a two-step decoding process. The key enzyme in this process, amino-acyl-tRNA synthetase must correctly couple a specific amino acid to its corresponding tRNA. Each one of 20 different synthetases recognizes one unique amino acid and all of its compatible or cognate tRNAs. Does a synthetase occasionally make a mistake and attach the wrong amino acid? Yes, some amino acids are very similar to others structurally, but if a mistake is made, the synthetase has an 'editing' mechanism which checks the fit of the aa-tRNA while still in its binding pocket and reverses the linking reaction if the shape of the product is not correct.
4. Figure 4-22, Lodish5e: Structure of tRNA. All tRNAs have two functions: 1) they are chemically linked to a specific amino acid and 2) they base-pair with a codon in mRNA, allowing the amino acid to be added to a growing polypeptide chain. A)The so-called cloverleaf diagram showing the four base-paired stems and three loops present in all tRNAs. But this is not the shape revealed by the molecular structure of tRNA. B) Computer-generated model of the folded phosphodiester backbone based on the solved molecular structure of crystallized tRNA.
5. Figure 4-23, Lodish5e: Nonstandard codon-anticodon base-pairing at the wobble position. The first and second bases in an mRNA codon form Watson-Crick (standard) base pairs with the third and second bases, respectively, of a tRNA anticodon. Surprisingly, bases in the third position, called the wobble position, can form a nonstandard base pair with the base in the first or wobble position of a tRNA anticodon. This explains how some cells can contain fewer than 61 tRNAs and still manage protein synthesis. A single tRNA anticodon can recognize more than one codon for a particular amino acid.
6. Figure 4-28, Lodish4e: Nonstandard wobble base-pairs. The G-U base pair fits into the DNA double helix almost as well as the standard Watson-Crick pair G-C. Inosine (I) can pair with A,C and U, so tRNA (3'-GAI-5') can recognize 4 of the 6 codons for leucine.
7. Figure 4-29, Lodish4e: Aminoacylation of tRNA. Amino acids are covalently linked to tRNAs by aminoacyl-tRNA synthetases. The aminoacylation reaction is shown here as a two-step process, both parts of which occur on the synthetase. All tRNAs have the same sequence CCA at their 3' ends, and this is where the amino acid is added. The amino acid must be 'activated' and ATP provides the activation energy. A complex of AMP-aa forms as an intermediate and the released PPi (pyrophosphate) is hydrolyzed by the enzyme pyrophosphatase with the release of energy to help make the overall reaction pathway more thermodynamically favorable. The aa is then transferred from its AMP carrier to the 3' terminal adenosine. Synthetases are divided into two classes depending on whether they add the amino acid to the 2' hydroxyl group (Class I) or the 3' hydroxyl group (Class II) of the ribose moiety of adenosine.
8. Figure 4-30, Lodish4e: Recognition of a tRNA by aminoacyl synthetases. The three-dimensional structures of two different classes of synthetases are shown in outline in this figure. The tRNA (a modified form of asparagine tRNA is shown as a ribbon diagram between them. This tRNA can bind to either class of synthetase. The blue balls in the tRNA represent nucleotides that contact the surface of class II AspRS synthetase whereas the yellow balls represent nucleotides that contact the surface of class I ArgRS synthetase. As the synthetases move close to the tRNA for binding, the complementary surface interactions become very obvious. The significance of the two classes of synthetases in not clear. The class I synthetases transfer the aminoacyl group to the 2' hydroxyl of the terminal adenosine whereas class II synthetases transfer the aminoacyl group to the 3' hydroxyl of the terminal adenosine.
9. Figure 4-24, Lodish5e: The general structure of ribosomes in prokaryotes and eukaryotes. Each ribosome consists of a large and a small subunit. All ribosomes contain two main rRNA molecules. They can be described by size as 23S and 16S rRNA in bacteria (S stands for Svedberg, a unit of centrifugation related to size), and 28S and 18S rRNA in vertebrate cells. There is also a smaller 5S rRNA in both. The 60S large subunit of vertebrate ribosomes also has a 5.8S rRNA based-paired to the 28S rRNA. The two ribosomal subunits of bacteria contain different sets of proteins, as do the two vertebrate ribosomal subunits.
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