Question

In this chapter, we focused on the translation of mRNA into proteins as well as on protein structure and function. Along the way, we found many opportunities to consider the methods and reasoning by which much of this information was acquired. From the explanations in the chapter, what answers would you propose to the following fundamental questions: (a) What experimentally derived information led to Holley's proposal of the two-dimensional cloverleaf model of tRNA? (b) What experimental information verifies that certain codons in mRNA specify chain termination during translation? (c) How do we know, based on studies of Neurospora nutritional mutations, that one gene specifies one enzyme? (d) On what basis have we concluded that proteins are the end products of genetic expression? (e) How do we know that the structure of a protein is intimately related to the function of that protein?

Short answer

Answer: Holley's cloverleaf model of tRNA and the one-gene-one-enzyme hypothesis significantly contributed to our understanding of genetic expression and protein synthesis. The cloverleaf model provided insight into the structure and function of tRNA molecules, which play an essential role in translation, the process by which the genetic code in mRNA is used to synthesize proteins. The one-gene-one-enzyme hypothesis highlighted the concept that individual genes are responsible for encoding specific enzymes or proteins, establishing a foundational concept in molecular biology linking genes to the proteins they produce and their functions within cells. Both discoveries are crucial to our comprehension of the steps involved in converting genetic information into functional protein molecules.

Step-by-step solution

(a) Holley's proposal of the cloverleaf model of tRNA.

In order to propose the two-dimensional cloverleaf model of tRNA, Robert Holley and his colleagues used the techniques of partial hydrolysis, gel electrophoresis, and chromatography to analyze the nucleotide sequence of each tRNA molecule. Their approach was to break the molecule into smaller fragments and separate them by size or charge, then determine the sequence within the fragments, and finally piece the overlapping fragments back together to obtain the full sequence. This led to Holley's observation that tRNA molecules have an internal structure with loops and a stem, which he described as a cloverleaf shape.

(b) Experimental verification of chain termination codons.

Several experiments were conducted to identify codons that specify chain termination during translation. One key experiment in this area was conducted by Marshall Nirenberg and Philip Leder, in which they used a cell-free protein-synthesizing system and synthetic mRNA containing only a single nucleotide base (for instance, only UUU repeats). By observing which amino acids were incorporated or not incorporated into the synthesized polypeptides, they were able to deduce the corresponding codons for each amino acid, and also identify the codons that specify chain termination (known as stop codons). These stop codons, UAA, UAG, and UGA, were found to terminate translation, releasing the polypeptide from the ribosome.

(c) One gene specifies one enzyme hypothesis.

The "one gene, one enzyme" hypothesis was first proposed by George Beadle and Edward Tatum after their studies on the fungus Neurospora crassa. By exposing the fungus to X-rays, they were able to induce mutations that affected the biosynthesis of certain amino acids. They found that these mutations affected specific steps in the synthesis pathway, leading to the accumulation of precursors and the absence of the end product (the amino acid). This suggested that a single gene was responsible for encoding an enzyme that catalyzes a specific reaction in the synthesis pathway. Further studies using similar reasoning and experimental methods reinforced the "one gene, one enzyme" hypothesis and later expanded it to "one gene, one protein" since proteins other than enzymes were also found to be encoded by individual genes.

(d) Proteins as end products of genetic expression.

Numerous experiments conducted on characterizing gene expression pathways have provided evidence that proteins are the end products of genetic expression. A series of steps, including transcription of DNA into mRNA, translation of mRNA into a polypeptide chain, and subsequent folding, modifications, and assembly of the polypeptide into a functional protein, lead to the production of a protein. The identification of specific gene sequences in DNA and their corresponding protein products, as well as the detailed molecular mechanisms governing these steps, led to the conclusion that proteins are the end products of genetic expression.

(e) Protein structure and function relationship.

There is a wealth of experimental evidence supporting the idea that protein structure is intimately related to its function. The primary structure, or amino acid sequence, of a protein determines the secondary and tertiary structures (e.g., alpha-helices, beta-sheets, and their overall three-dimensional folding), which ultimately determine the quaternary structure (the interaction between multiple protein subunits) in some cases. The structure of a protein directly affects its biochemical properties, such as its ability to bind substrates or ligands, its stability in various cellular environments, and its susceptibility to degradation. Additionally, structural studies of enzymes and other proteins, often done using techniques like X-ray crystallography or NMR spectroscopy, provide insights into their mechanisms of action and how mutations in their structure can lead to diseases or other functional defects.

Key concepts

tRNA Cloverleaf Model

Understanding the intricate design of the transfer RNA (tRNA) molecule is crucial when diving into the molecular biology realms. The tRNA cloverleaf model, proposed by Robert Holley, provides a visual depiction of tRNA's structure. This model came to life thanks to meticulous analysis involving techniques like partial hydrolysis, gel electrophoresis, and chromatography.

The cloverleaf arrangement showcases four arms—each with a specific role in protein synthesis. There's the acceptor stem for amino acid attachment, the TψC arm interacting with ribosomes, the anticodon arm pairing with messenger RNA (mRNA), and the DHU arm for tRNA stability. Each arm forms by the base pairing of nucleotides, creating the tRNA's secondary structure. The importance of this model lies in its role in translation, the process where the genetic code carried by mRNA is transformed into a protein, with tRNA as a key interpreter.

Chain Termination Codons

Chain termination codons, or 'stop codons', are essentially the full stops in the language of genetic code. These unique triplets within mRNA—UAA, UAG, and UGA—do not correspond to any amino acid. Rather, they signal the end of protein synthesis. The discovery of these codons was pivotal, as elucidated by experiments like those conducted by Marshall Nirenberg and Philip Leder using synthetic mRNAs and a cell-free system.

When the translation machinery of a cell encounters these stop codons during mRNA reading, release factors trigger the release of the newly formed polypeptide. Therefore, these termination signals ensure proteins are synthesized to the correct length, maintaining their intended function.

One Gene One Enzyme Hypothesis

The one gene, one enzyme hypothesis is a foundational concept demonstrating the precise nature of gene function. George Beadle and Edward Tatum's experiments using the bread mold Neurospora crassa served as a stepping stone to this discovery. The duo irradiated the fungus, causing mutations that resulted in the loss of specific enzymes for amino acid synthesis.

This led to the hypothesis that each gene is responsible for the production of a single enzyme that in turn affects a particular metabolic pathway. Over time, this hypothesis evolved to

Protein Structure-Function Relationship

The intricate relationship between a protein's structure and its function is like a lock and key mechanism where every detail matters. Primary structure, determined by amino acid sequence, dictates the protein's folding pattern into secondary and tertiary configurations. This folding is vital, as it influences the enzyme's biochemical activity, stability, and functionality within the cell.

X-ray crystallography and NMR spectroscopy have provided a window into understanding how proteins work. These methods have shown that even slight alterations in structure due to mutations can have significant impacts on a protein's function, affecting health and disease. Thus, the 3D shape of proteins is not only indicative of their roles but is also critical in bioengineering, drug design, and understanding disease pathology.