Question

Three fragments are produced when a piece of RNA is treated with pancreatic RNase. According to the amount of phosphate, these fragments are highly acidic (much phosphate), moderately acidic, and slightly acidic (little phosphate). Each of these fragments is further hydrolyzed with \(\mathrm{NaOH}\). The resulting nucelotides are: 1\. highly acidic fragment: Gp, \(\mathrm{pAp}, \mathrm{Cp}\) 2\. moderately acidic fragment: Gp, \(\mathrm{Up}\) 3\. slightly acidic fragment: G, Gp What is the sequence of this piece of RNA?

Short answer

The sequence of the RNA piece is \[\text{5'} - GpApCGpUpG - \text{3'}.\]

Step-by-step solution

Reversing the Hydrolysis with NaOH

After hydrolyzing with NaOH, we have the following nucleotides for each fragment: 1. highly acidic fragment: Gp, pAp, Cp 2. moderately acidic fragment: Gp, Up 3. slightly acidic fragment: G, Gp Let's reverse the hydrolysis by NaOH - for each fragment, we connect the nucleotides with 3'-5' phosphodiester bonds (#p# indicates a phosphate group). 1. highly acidic fragment: GpApC 2. moderately acidic fragment: GpU 3. slightly acidic fragment: GpG

Finding the Common Links

We have to find the appropriate way to connect the fragments to form the original RNA sequence. As we can see, there is a common nucleotide between: - highly acidic and moderately acidic fragment: G - moderately acidic and slightly acidic fragment: Gp

Forming the RNA Sequence

Now that we've found the common links, we can connect our fragments in the following manner: 1. Connect the highly acidic and moderately acidic fragment with G: GpApC - G - pU (notice the common link G in both fragments) 2. Connect the moderately acidic and slightly acidic fragment with Gp: GpApCGp - G - pG (notice the common link Gp in both fragments) So, our final RNA sequence is: \[\text{5'} - GpApCGpUpG - \text{3'}\]

Key concepts

Nucleotide Hydrolysis

The process of nucleotide hydrolysis involves breaking down nucleotides into their components, which often includes the removal of phosphate groups. A nucleotide consists of a nitrogenous base, a sugar molecule, and one or more phosphate groups. During hydrolysis, particularly in the presence of strong bases like NaOH, the phosphodiester bonds that connect nucleotides in RNA can be broken.
This leads to the release of individual nucleotides. Depending on how the nucleotides are released, they often carry different levels of acidity due to the number of phosphates present:

• Highly acidic, with more phosphates.
• Moderately acidic, with fewer phosphates.
• Slightly acidic, with minimal or no phosphates.

Understanding nucleotide hydrolysis is key for interpreting the fragmentation patterns that arise in enzymatic digestion of RNA, such as the exercise involving pancreatic RNase. It allows us to deduce the sequence of an RNA strand by mapping back the fragments produced.

Phosphodiester Bonds

Phosphodiester bonds are the chemical bonds that form the backbone of RNA molecules. These bonds connect the 3’-hydroxyl group of one nucleotide’s sugar molecule to the 5’-phosphate group of the next nucleotide. The resulting chain is known as a **polynucleotide chain**. The sequence and integrity of phosphodiester bonds are crucial.
They define the overall structure and proper functioning of the RNA molecule. During processes like nucleotide hydrolysis and RNA sequencing, understanding these bonds is essential. If these bonds are broken, as seen in nucleotide hydrolysis, identifying how they originally connected helps reconstruct the RNA sequence.
The sequential alignment of nucleotides—connected via 3'-5' phosphodiester bonds—translates into the RNA strand running from the 5’ end to the 3’ end. Manipulating these bonds in a controlled manner like in laboratory settings, reveals structural insights into RNA biology.

RNA Structure

The structure of RNA is central to its function in biological systems. RNA comprises a single-stranded polynucleotide chain consisting of nucleotides. Each nucleotide carries one of four nitrogenous bases: adenine (A), guanine (G), cytosine (C), or uracil (U). These bases engage in various interactions; most commonly, they form hydrogen bonds with complementary bases to help stabilize the structure in certain functions.
The flexibility of its single strand allows RNA to fold into complex three-dimensional shapes critical to its role in protein synthesis and other cellular processes. From a structural standpoint, stacking interactions, while not bonds, also help stabilize RNA due to the planar nature of the nucleotide bases, promoting hydrophobic interactions.

• RNA's adaptability provides a diverse range of functions, from coding, decoding, regulation, and expression of genes, to catalysis as seen in ribozymes.
• Understanding RNA structure lays the groundwork for biochemistry, genetics, and molecular biology.

Knowledge of RNA structure is beneficial when analyzing experiments involving RNA digestion and reconstruction, like determining sequences from fragmented samples.

Pancreatic RNase

Pancreatic RNase, or ribonuclease A, is an enzyme responsible for digesting RNA. It cleaves the phosphodiester bonds within RNA, primarily targeting sites adjacent to pyrimidine bases (cytosine and uracil). This specific cleavage results in the production of RNA fragments that can then be analyzed further.
This enzyme plays a vital research role by enabling scientists to dissect RNA sequences into more manageable parts for analysis, particularly for sequencing and structural studies. Additionally, pancreatic RNase’s activity provides insights into understanding RNA stability and degradation processes in both physiological and experimental contexts. Its precise cleavage pattern helps predict the nature of fragmented ends from digested RNA—whether the fragments are cyclic or linear—which aids in reconstructing the original RNA strand.

• The enzyme's widespread use in biochemistry demonstrates the significance of enzymatic digestion for studying complex biomolecules like RNA.
• Effective use of pancreatic RNase showcases how enzymes can be utilized to yield detailed and accurate biochemical information.

By understanding the action of pancreatic RNase, we can recreate the sequence of an RNA molecule, thus providing insights into its original structural and functional roles.