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 explana- tions given in the chapter, what answers would you propose to the following fundamental questions: (a) Why did geneticists believe, even before direct experimental evidence was obtained, that the genetic code would turn out to be composed of triplet sequences and be nonoverlapping? Experimentally, how were these suppositions shown to be correct? (b) What experimental evidence provided the initial insights into the compositions of codons encoding specific amino acids? (c) How were the specific sequences of triplet codes determined experimentally? (d) How were the experimentally derived triplet codon assignments verified in studies using bacteriophage MS2?

Short answer

Answer: Geneticists believed in the triplet and nonoverlapping nature of the genetic code because a triplet code provides enough combinations (64) to specify all 20 amino acids used in proteins. A nonoverlapping code allows unambiguous reading, with no base being part of more than one codon. Experiments that supported these ideas included Crick, Brenner, Barnett, and Watts-Tobin's work with T4 phage mutations, and Nirenberg and Matthaei's cell-free in vitro translation system, which confirmed the triplet sequences encoding specific amino acids.

Step-by-step solution

(a) Belief in Triplet and Nonoverlapping Genetic Code)

Geneticists believed in the triplet and nonoverlapping nature of the genetic code because of the following reasons: 1. A triplet code has enough combinations (4^3 = 64) to specify all 20 amino acids used in proteins. 2. A doublet code (4^2 = 16) would not have enough combinations and a quadruplet code (4^4 = 256) would have too many. 3. To allow unambiguous reading, the genetic code should be nonoverlapping, which means that no base is part of more than one codon or shared between codons. The experiments that proved these ideas include: 1. Crick, Brenner, Barnett, and Watts-Tobin (1961) used mutations in T4 phage, which suggested that the code is read in a non-overlapping way. 2. Nirenberg and Matthaei's cell-free in vitro translation system confirmed that the code is read in triplet sequences encoding specific amino acids.

(b) Initial Insights into Codon Compositions)

Initial insights into the compositions of codons encoding specific amino acids came from the experiments conducted by Nirenberg and Matthaei (1961). They used a cell-free in vitro translation system with synthetic RNAs as a template containing only one type of ribonucleotide. They discovered that poly(U) RNA coded for the amino acid phenylalanine, revealing the existence of a codon-anticodon interaction.

(c) Determining Specific Triplet Code Sequences)

The specific sequences of triplet codes were determined experimentally using the following techniques: 1. Nirenberg and Leder (1964) developed the triplet binding assay, in which synthetic triplet RNAs were mixed with ribosomes and radioactively labeled tRNAs to identify which triplet bound to which tRNA carrying an amino acid. 2. Khorana and colleagues synthesized various combinations of dinucleotide and trinucleotide repeats, establishing several specific triplet codon assignments for different amino acids. These experiments resulted in the "codon table," which lists all 64 triplet codons and the amino acids they specify.

(d) Verifying Triplet Codon Assignments with Bacteriophage MS2)

The experimentally derived triplet codon assignments were verified in studies using bacteriophage MS2 by comparing the amino acid sequences of the proteins produced using the known nucleotide sequence of MS2 RNA. These studies further confirmed the universality and specificity of the genetic code as well as the validity of the codon table, as the experimentally determined codon assignments matched the amino acid sequences of bacteriophage MS2 proteins.

Key concepts

Transcription: Copying DNA to RNA

Picture transcription as the meticulous process of a scribe copying a crucial manuscript. In molecular biology, it's the first step in expressing genetic information. Transcription specifically refers to the synthesis of RNA from a DNA template.

During transcription, enzymes called RNA polymerases bind to DNA at a sequence called the promoter. They then uncoil the DNA and assemble a strand of messenger RNA (mRNA) that mirrors the sequence of the DNA template, except for the substitution of the base uracil (U) for thymine (T).

This mRNA strand then carries the genetic message from the DNA out of the nucleus and into the cytoplasm, where it will provide instructions for building proteins during translation. Given its critical role in gene expression, understanding transcription is fundamental to grasping the broader concepts of genetics and molecular biology.

Triplet Sequences: The DNA Code Words

Consider triplet sequences as unique three-letter words in a language that translate to specific meanings—in this case, amino acids. These triplets, known as codons, are sequential groups of three nucleotide bases in the DNA or RNA that specify which amino acid will be added next during protein synthesis.

An array of these triplets forms a gene, a series of instructions for building specific proteins. It's the specific order of these triplets that encodes the precise instructions for the amino acid sequence of a protein, leading to the vast diversity of proteins possible for cellular functions.

The rationale for believing in triplet sequences was due in part to simple mathematics—three nucleotide bases allow for 64 unique combinations, enough to code for all 20 amino acids along with signals to start and stop protein synthesis, a concept elegantly simple yet crucial for proper genetic translation.

Codon-Anticodon Interaction: The Genetic Conversation

Imagine a lock and key mechanism, where only the right key can open a specific lock. The codon-anticodon interaction works similarly. A codon on the mRNA is a three-base sequence that specifies an amino acid. The corresponding key is an anticodon, a three-base sequence on a molecule of transfer RNA (tRNA) that is complementary to the codon.

When the tRNA carrying an amino acid recognizes and pairs with the matching codon on the mRNA strand through base-pairing rules, the amino acid gets added to the growing protein chain. This precise interaction ensures that the genetic code translates correctly into functional proteins, and is a cornerstone of the translation process that converts genetic information into life-sustaining chemical actions.

Cell-Free In Vitro Translation: A Genetic Test Tube

To understand complex biological processes, scientists often recreate these processes outside of living organisms, in a controlled test tube environment—known as in vitro. Cell-free in vitro translation is one such powerful technique where cellular components necessary for protein synthesis are extracted and the process of translation is carried out in a test tube.

This experimental set-up allows scientists to study the mechanics of protein synthesis, examine the effects of various RNA sequences on the production of proteins, and determine the composition of codons. Historical experiments using cell-free in vitro translation systems greatly contributed to decoding the genetic code and confirming the triplet nature of codons.

Triplet Binding Assay: Deciphering the Genetic Code

Imagine a detective examining evidence to identify a suspect. The triplet binding assay was a scientific detective work to decipher the genetic code. In this assay, researchers used ribosomes, radioactively labeled tRNAs, and synthetic RNAs with known sequences to find out which amino acid corresponded to which three-base RNA sequence (triplet).

By observing which synthetic triplet RNA sequences bound to which tRNAs, they could begin to map the genetic code, determining the specific codons that correspond to each amino acid. This assay was integral to piecing together the codon table, a critical key to understanding genetic code and how it directs protein synthesis.