Question

In this chapter, we focused on the genetic code and the transcription of genetic information stored in DNA into complementary RNA molecules. Along the way, we found many opportunities to consider the methods and reasoning by which much of this information was acquired. From the explanations given in the chapter, what answers would you propose to the following fundamental questions: (a) How did we determine the compositions of codons encoding specific amino acids? (b) How were the specific sequences of triplet codes determined experimentally? (c) How were the experimentally derived triplet codon assignments verified in studies using bacteriophage MS2? (d) How do we know that mRNA exists and serves as an intermediate between information encoded in DNA and its concomitant gene product? (e) How do we know that the initial transcript of a eukaryotic gene contains noncoding sequences that must be removed before accurate translation into proteins can occur?

Short answer

Answer: Nirenberg and Matthaei synthesized artificial RNA molecules with repeating nucleotide sequences, such as poly-U (UUU…), and added them to an in vitro translation system. The amino acid sequence of the resulting polypeptides was then determined, revealing the corresponding codons for each amino acid. For example, poly-U mRNA directed the synthesis of a polypeptide containing the amino acid phenylalanine, establishing that the triplet code UUU codes for phenylalanine.

Step-by-step solution

a) Codons encoding specific amino acids

To determine the compositions of codons encoding specific amino acids, biochemists Marshall Nirenberg and Heinrich Matthaei performed an experiment in the 1960s. They synthesized artificial RNA molecules with repeating nucleotide sequences, such as poly-U (UUU…). These repeating sequences were added to an in vitro translation system, which then produced polypeptides consisting of repeating amino acids. The amino acid sequence of these polypeptides was then determined, revealing the corresponding codons for each amino acid. For example, poly-U mRNA directed the synthesis of a polypeptide containing the amino acid phenylalanine, thus establishing that the triplet code UUU codes for phenylalanine.

b) Triplet codes determined experimentally

To determine the specific sequences of triplet codes, scientists used in vitro translation systems and synthetic RNA molecules with known sequences. They systematically tested different triplet combinations to synthesize specific amino acids. To avoid ambiguities, they used triplet-binding assays, which monitored the binding of specific triplet nucleotide sequences to aminoacyl-tRNA molecules. This experimental approach allowed them to establish the sequences of each of the 64 triplet codons, assigning them to one of the 20 amino acids or the stop signals.

c) Verifying triplet codon assignments with bacteriophage MS2

To verify the experimentally derived triplet codon assignments, researchers used bacteriophage MS2 as a model system. Bacteriophage MS2 is a small virus that infects bacteria, and its genome consists of a single-stranded RNA molecule. By determining the nucleotide sequence of this RNA molecule and comparing it to the experimentally derived genetic code, researchers were able to verify the accuracy of the codon assignments. They experimentally mutated specific codons in the bacteriophage MS2 genome and observed the corresponding changes in the amino acid sequence of the virus' proteins, confirming the codon assignments.

d) Existence and role of mRNA

The existence of mRNA and its role as an intermediate between DNA and its corresponding gene product were first demonstrated by the experiments of Francois Jacob, Sydney Brenner, and Matthew Meselson. They used a bacterium infected with a bacteriophage and labeled the newly synthesized RNA with radioactive isotopes. By tracking the decay of radioactivity in the cytoplasm and protein of the bacteria, they demonstrated that a new RNA species carries genetic information from DNA to the site of protein synthesis. Further experiments showed that this RNA molecule is complementary to the DNA molecule, indicating that it is synthesized from DNA through the process of transcription.

e) Noncoding sequences in eukaryotic gene transcripts

The presence of noncoding sequences in eukaryotic gene transcripts was discovered through studies on the complete nucleotide sequences of eukaryotic genes and their corresponding transcripts. Researchers observed that the initial transcripts contained sequences that were not present in the final mRNA molecules and did not code for amino acids. These extra sequences, called introns, need to be removed through a process called splicing before the mRNA molecule is translated into a protein. Researchers confirmed the presence of introns by comparing the DNA sequences of eukaryotic genes with the amino acid sequences of their corresponding proteins and their mature mRNA molecules. This finding led to the understanding that eukaryotic genes contain noncoding sequences which must be removed before accurate translation can occur.