Question

Even though some students correctly synthesize a fluorescent cDNA probe complementary to mRNA for a particular yeast protein, they find that the probe does not attach to any portion of the yeast's genome. Explain why the students' probe does not work.

Short answer

The students' cDNA probe does not work because it is derived from mRNA which only includes exons, the coding sequences. But the yeast's genomic DNA contains both exons and introns. Introns are non-coding sequences that get spliced out during the creation of mature mRNA in eukaryotic cells. Therefore, the cDNA probe, which is intron-less, won't bind to the intron-containing genomic DNA.

Step-by-step solution

Understanding DNA transcription

Transcription is the first step of gene expression. It's a process where a segment of DNA is copied into RNA by the enzyme RNA polymerase. These mRNA molecules contain sections called exons (coding regions) and introns (non-coding regions). After transcription and before leaving the nucleus, the mRNA goes through splicing where the introns are removed and exons are joined.

Understanding cDNA Synthesis

cDNA synthesis is typically performed through reverse transcription of mRNA by using reverse transcriptase enzyme. The cDNA is thus complementary to the mRNA, meaning it corresponds to a protein coding sequence (exons only), since introns are removed during mRNA splicing. Therefore, cDNA doesn't contain sequences that are complementary to any introns present in the DNA.

Why the probe doesn't work

The students' cDNA probe is complementary to spliced mRNA which only includes exons, the coding sequences. It will not bind to intron sequences in the genomic DNA. Therefore, if the probe doesn't bind anywhere on the yeast genome, it suggests that the mRNA from which the cDNA probe was derived includes spliced-out introns which are not present in the same configuration in the genomic DNA.

Key concepts

mRNA Splicing

mRNA splicing is a crucial process that takes place during the formation of mature mRNA from precursor mRNA, also known as pre-mRNA. Initially, when DNA is transcribed into RNA, the resultant RNA strand, or pre-mRNA, contains both exons and introns. Exons are sequences that code for proteins, and introns are non-coding sequences.
To make the mRNA functional, it undergoes splicing in the nucleus, where introns are removed, and exons are joined together. This process is essential because the remaining exons form a continuous protein-coding sequence that can be translated effectively during protein synthesis.

• Splicing occurs in a protein-RNA complex called the spliceosome.
• After splicing, the mRNA is mature and ready to leave the nucleus for translation into protein.
• This mature mRNA is what will be reverse transcribed into cDNA.

The removal of introns and joining of exons ensures that the mRNA is able to direct the synthesis of proteins correctly.

DNA Transcription

DNA transcription is the initial step in the journey of going from a gene to a protein. In this process, DNA is used as a template to synthesize RNA, specifically mRNA, which will then serve as a blueprint for protein synthesis.
During transcription, the enzyme RNA polymerase travels along the DNA strand, unzipping the double helix and synthesizing a single strand of RNA by matching complementary nucleotides to the DNA template. Notably, the RNA sequence produced contains both coding (exons) and non-coding (introns) regions.

• This process happens in the nucleus of eukaryotic cells.
• It results in the formation of pre-mRNA, which must be processed before it can be translated into protein.
• The transcription process is closely regulated to ensure genes are expressed at the right time and at appropriate levels.

DNA transcription is an essential step for gene expression as it bridges the gap between the genome and proteome.

Reverse Transcription

Reverse transcription is a fascinating process that turns RNA molecules back into DNA. This is the opposite of the usual process of transcription, and it is carried out by an enzyme called reverse transcriptase.
This process is particularly important in the synthesis of cDNA (complementary DNA). When scientists want to create a cDNA probe for laboratory experiments, they use reverse transcription to produce a DNA version of the mRNA molecule.

• cDNA synthesis is crucial in studying genes and gene expression.
• Since mRNA is already spliced, the resulting cDNA consists only of sequences corresponding to exons.
• cDNA lacks introns, unlike most nuclear DNA, which can lead to mismatches when comparing genomic DNA to a cDNA probe.

This process provides a valuable tool for various genetic engineering and molecular biology techniques.

Gene Expression

Gene expression is the process through which information from a gene is used to construct a functional product, typically a protein. This intricate process involves multiple steps, beginning with DNA transcription and followed by mRNA processing, mRNA splicing, translation, and post-translational modifications.
In eukaryotic cells, gene expression is a tightly regulated phenomenon, ensuring that the right genes are expressed at the correct times to maintain normal cellular functions. This regulation happens at various stages:

• Transcriptional Control: Determines when and how fast a gene is transcribed.
• RNA Processing Control: Regulates the modification of the newly synthesized RNA.
• Translational Control: Influences how efficiently mRNA is translated into proteins.
• Post-Translational Control: Modifies the activity and stability of the resulting proteins.

Understanding gene expression is fundamental for deciphering cellular behavior, developmental biology, and many health-related conditions.