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) 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? (e) What evidence do we have that the expression of the information encoded in DNA involves an RNA intermediate? (f) 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

During the early stages of molecular biology, geneticists deduced that the genetic code must be composed of triplet sequences and be nonoverlapping for two main reasons. Firstly, a triplet sequence provides 64 possible combinations, which is more than enough to code for 20 amino acids, while a single or double base system would not suffice. Secondly, nonoverlapping codes were suggested to minimize errors in translation and maintain the integrity of genetic information. These suppositions were experimentally shown to be correct through the work of Crick and others using frame-shift mutations. Question (b): What was the experimental evidence used to determine the composition of codons encoding specific amino acids? Experimental evidence for the composition of codons encoding specific amino acids came from the work of Nirenberg and Matthaei, who used synthetic RNA molecules in a cell-free system. By using different RNA sequences, they were able to deduce the amino acids that would be incorporated into the synthesized polypeptides. This allowed them to make correlations between specific RNA triplet sequences (codons) and the corresponding amino acids they encoded. Question (c): How were the specific sequences of triplet codes experimentally determined? The specific sequences of triplet codes were experimentally determined through RNA homopolymers, RNA heteropolymers, and in vitro protein synthesis using synthetic trinucleotides. Researchers systematically controlled the sequences in the synthesized RNA and analyzed the resulting protein products to deduce the triplet codon sequences for each of the 20 amino acids. Question (d): How were the experimentally derived triplet codon assignments verified? The experimentally derived triplet codon assignments were verified using bacteriophage MS2. By sequencing the RNA of bacteriophage MS2 and comparing the predicted protein products based on the known triplet codon assignments with the actual protein products synthesized by the virus, researchers were able to confirm the validity of the triplet codon assignments. Question (e): What evidence supports the involvement of RNA intermediates in the expression of genetic information? The evidence for the expression of information encoded in DNA involving an RNA intermediate comes from multiple sources, such as the "Central Dogma of Molecular Biology," which states that information flows from DNA to RNA via transcription, and from RNA to proteins via translation. Additionally, the presence and roles of various RNA molecules, such as mRNA, tRNA, and rRNA, in protein synthesis provide indirect evidence for the involvement of an RNA intermediate. Question (f): Why are there noncoding sequences in eukaryotic gene transcripts, and how were they discovered? Noncoding sequences, called introns, are present in eukaryotic gene transcripts and must be removed before accurate translation into proteins can occur. Introns were discovered through the work of several researchers, such as Sharp and Roberts, who demonstrated the discontinuity of eukaryotic genes through electron microscopy and RNA-DNA hybridization experiments. Introns are removed through a process called splicing, which results in the mature mRNA molecule containing only the coding regions (exons).

Step-by-step solution

Question (a): Genetic Code is Triplet and Nonoverlapping

During the early stages of molecular biology, geneticists deduced that the genetic code must be composed of triplet sequences and be nonoverlapping for the following reasons: 1. The basic unit of genetic information should have the ability to code for all possible amino acids. Since there are 20 amino acids and only 4 types of DNA bases (A, C, G, and T), a single base would be insufficient to code for all amino acids. A doublet sequence would only provide 4^2 = 16 combinations. A triplet sequence has 4^3 = 64 possibilities, which is more than enough to code for 20 amino acids. 2. The nonoverlapping property was suggested to minimize errors in translation and maintain the integrity of genetic information. Overlapping codes could cause multiple amino acid errors in translation if a single nucleotide is mutated, while nonoverlapping codes would only affect one amino acid. These suppositions were experimentally shown to be correct through the work of Crick and others using frame-shift mutations, which involved adding or subtracting base pairs from the coding sequence. When a single or a pair of base pairs was added, the translation was significantly impacted, causing multiple errors in the amino acid sequence. However, when three base pairs were added or subtracted, the translation process was minimally affected, and only a single amino acid error occurred. This provided evidence for the triplet and nonoverlapping genetic code.

Question (b): Experimental Evidence for Codon Composition

The experimental evidence for the composition of codons encoding specific amino acids came from the work of Nirenberg and Matthaei, who used synthetic RNA molecules to decipher the genetic code. By using different RNA sequences in a cell-free system, they were able to deduce the amino acids that would be incorporated into the synthesized polypeptides. This allowed them to make correlations between specific RNA triplet sequences (codons) and the corresponding amino acids they encoded.

Question (c): Determination of Triplet Code Sequences

The specific sequences of triplet codes were experimentally determined through RNA homopolymers, RNA heteropolymers, and in vitro protein synthesis using synthetic trinucleotides. These experiments, conducted by scientists such as Nirenberg, Khorana, and Holley, involved synthesizing RNA molecules with different base sequences and observing which amino acids were incorporated into the synthesized proteins. By systematically controlling the sequences in the synthesized RNA and analyzing the resulting protein products, the researchers were able to deduce the triplet codon sequences for each of the 20 amino acids.

Question (d): Verification of Triplet Codon Assignments

The experimentally derived triplet codon assignments were verified using bacteriophage MS2, a bacterial virus possessing a single-stranded RNA genome. By sequencing the RNA of bacteriophage MS2 and comparing the predicted protein products based on the known triplet codon assignments with the actual protein products synthesized by the virus, researchers were able to confirm the validity of the triplet codon assignments.

Question (e): Evidence for RNA Intermediate

The evidence for the expression of information encoded in DNA involving an RNA intermediate comes from multiple sources. One key piece of evidence is the "Central Dogma of Molecular Biology," which states that information flows from DNA to RNA via transcription, and from RNA to proteins via translation. Additionally, the presence of various types of RNA molecules, such as mRNA, tRNA, and rRNA, and their roles in protein synthesis provide indirect evidence for the involvement of an RNA intermediate in the expression of genetic information.

Question (f): Presence of Noncoding Sequences in Eukaryotic Gene Transcripts

We know that the initial transcript of a eukaryotic gene contains noncoding sequences, called introns, that must be removed before accurate translation into proteins can occur. This is due to experiments that have shown that eukaryotic genes are composed of alternating sections of coding (exons) and noncoding (introns) regions. Following transcription, these introns must be removed through a process called splicing, which results in the mature mRNA molecule containing only the coding regions (exons). The presence of introns in eukaryotic gene transcripts was discovered through the work of several researchers, including Sharp and Roberts, who demonstrated the discontinuity of eukaryotic genes through electron microscopy and RNA-DNA hybridization experiments.

Key concepts

Triplet Sequences

In the genetic code, the information needed to synthesize proteins is stored in triplet sequences known as codons. Each triplet consists of three nucleotide bases. With four different nucleotides (A, C, G, T in DNA and A, C, G, U in RNA), these triplets provide 64 possible combinations (4^3). This is important because there are 20 standard amino acids that proteins are built from. The brilliance of triplet sequences is that they provide plenty of combinations to code for all these amino acids, with extra codons available for punctuation, like stop signals that tell the cellular machinery when to stop reading the code.

Thus, the genetic code has built-in redundancy; for example, several codons can code for the same amino acid. This redundancy acts as a buffer against some mutations, ensuring that despite genetic errors, the correct protein can still be made in many cases. This concept was crucial for geneticists as they embarked on deciphering the genetic language.

Nonoverlapping Code

The genetic code is described as nonoverlapping, which means that each nucleotide is part of only one codon, and codons are read one at a time without sharing nucleotides. This was important for genetic stability. If the code were overlapping, a single mutation could affect multiple codons, potentially causing widespread errors in the protein sequence.

Nonoverlapping sequences improve the efficiency and accuracy of protein synthesis. Mutations will generally only affect a single amino acid in the resulting protein, thus localizing the impact. Understanding nonoverlapping codes helped scientists understand how genetic material maintains integrity through cellular processes and environmental changes.

Experimental Validation of Codons

Experimental validation of codons was a cornerstone in molecular biology. Scientists like Nirenberg and Matthaei played a pivotal role in cracking the code. By using synthetic RNA sequences in vitro, they matched specific RNA triplets (codons) with their corresponding amino acids, discovering which amino acids correspond to specific codons.

This method involved introducing RNA sequences into a controlled environment where they could observe how different sequences influenced amino acid assembly into proteins. From such groundbreaking work, scientists learned which sequences corresponded to which amino acids, forming the basis of our understanding of protein synthesis.

• This facilitated the creation of the genetic dictionary.
• It provided insights into how genes dictate protein production.

Such experiments marked the beginning of a clearer understanding of the language of life.

RNA Intermediate

The concept of an RNA intermediate was revolutionary in understanding gene expression. RNA acts as a go-between in the process of creating proteins from DNA. The central dogma of molecular biology describes this flow of genetic information: it moves from DNA to RNA (through transcription) and then from RNA to protein (through translation).

RNA serves multiple roles, with mRNA (messenger RNA) being primarily responsible for conveying genetic information from DNA in the nucleus to ribosomes, the protein factories in the cell cytoplasm. This proves the existence of an RNA intermediate in the expression process. Discovery of various RNA types like tRNA (transfer RNA) and rRNA (ribosomal RNA) further substantiated the importance of RNA as not just a messenger, but as an active participant in protein synthesis.

• RNA intermediate enables genetic information to be expressed correctly.
• It acts as a critical control point in gene regulation.

Introns and Exons

In eukaryotic genes, DNA sequences are not exclusively coding. There are introns and exons. Exons are coding sequences that get translated into proteins, while introns are noncoding sections that must be removed through a process called splicing to produce mature mRNA.

This concept became apparent through electron microscopy studies and RNA-DNA hybridization techniques, revealing that the initial mRNA transcript is longer than the final mRNA used for protein synthesis. Spliceosomes, complex entities, facilitate the removal of introns. Understanding introns and exons was crucial for explaining why a single gene can lead to multiple proteins through alternative splicing.

• Introns can play regulatory roles in gene expression.
• They provide opportunities for genetic recombination, increasing genetic diversity.

Through better understanding of introns and exons, we grasped how genetic complexity is managed and maintained in eukaryotic organisms.