Biological Chemistry - Berezov T. T., Korovkin B. F. 1998

Chemistry of Nucleic Acids
Structure of Nucleic Acids
Primary Structure of Nucleic Acids

The primary Structure of Nucleic Acids refers to the order and sequence of mononucleotides within the polynucleotide chains of DNA and RNA. Such chains are stabilized by 3',5'-phosphodiester bonds. Because the molecular weights of Nucleic Acids vary across a wide range (from 2∙10⁴ to 10¹⁰—10¹¹), determining the Introduction/19.html">Primary Structure of all known RNAs, and especially DNAs, is extremely difficult. Nevertheless, all nucleic acids (more precisely, single-stranded nucleic acids) share the same type of bond—a 3',5'-phosphodiester bond between adjacent NUCLEOTIDES. This common structural framework can be represented as follows:

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It has been established that the hydroxyl groups at the 3' and 5' positions of the carbohydrate residues participate in The formation of the internucleotide bond.

To date, researchers have successfully determined the primary structure of almost all tRNAs, A number of 5S rRNA and 16S rRNA molecules of E. coli, and various viral RNAs comprising hundreds and thousands of nucleotide residues. An approximate scheme of The nucleotide sequence in an RNA molecule is given below. All cellular RNAs are essentially composed of a single-stranded polynucleotide chain:

The polynucleotide chain of an RNA molecule almost always possesses a free monophosphoric ester at one end, conventionally designated as the 5' end; at the opposite end of the chain, such a phosphate is absent, and instead a nucleotide with free 2' and 3' hydroxyl groups is present. If an RNA molecule is subjected to alkaline Hydrolysis, the terminal nucleotides recovered will be CMP with a free phosphate at the 5' end and free adenosine as a free nucleoside at the 3' end of the polynucleotide chain.

Stepwise hydrolysis Methods, carried out primarily by exonucleases and involving the sequential Cleavage of single mononucleotides from one end of the nucleic acid molecule, have played a decisive role in elucidating the primary structure of RNA. Below is the primary STRUCTURE OF THE first RNA consisting of 77 nucleotides, whose nucleotide sequence was deciphered in 1965 by R. Holley et al., namely Alanine tRNA:

In this structure, P represents a phosphate residue, ψ is pseudo-UMP, MeG is methylguanine, DiHU is dihydrouracil, DiMeG is dimethylguanine, and MeI is methylinosine.

Two significant Features of the primary structure of all tRNAs should be specifically highlighted. The first is that the 5' end is always guanylic (rarely cytidylic) acid bearing a free phosphate residue at C-5'. The second feature is the presence at the opposite end of the molecule of three mononucleotide residues with an identical sequence—CCA, where the adenylic acid residue contains a free 3'-OH group*.

* Other Structural Features of tRNA are discussed in Chapter 14.

Between these structures, all other nucleotide residues are arranged in a strictly defined sequence, among which minor nucleotides account for up to 10%. The polynucleotide chain of various tRNA types contains approximately 75 nucleotides.

Messenger (informational) RNAs belong to the most heterogeneous class of nucleic acids, differing in mass (see Table 3.1), structure, size, stability, and Functions. The primary function of mRNA is to transfer information from DNA (more precisely, from a Gene) to the protein-synthesizing System of the Cell. mRNA acts as a template and, consequently, determines the primary structure of the synthesized protein (for details, see Chapter 14). mRNAs possess a number of specific primary structural features; in particular, at the 5' end, they all contain a specific ribonucleotide sequence known as a cap. The first nucleotide is 7-methylguanosine triphosphate, which is attached to the 5' hydroxyl of the adjacent mononucleotide, represented by a 2'-O-methylpurine nucleotide. At the other, 3' end, most (though not all) mRNAs contain a polyadenylate sequence (poly-A) numbering from 150 to 200 nucleotides.

The Role of mRNA "capping" and "polyadenylation" in Protein Synthesis has not been fully elucidated. It is hypothesized that the cap is necessary for specific recognition during Translation, whereas the poly-A tail is assigned the role of stabilizing the entire mRNA molecule.

In recent years, the primary structure has been deciphered not only for low-molecular-weight 5S rRNAs of various Bacteria and 5.8S rRNAs of animal Cells, but also for high-molecular-weight 16S and 18S rRNAs, which contain up to 1200–1500 nucleotide units. Furthermore, The nucleotide sequences of E. coli 23S rRNA and Yeast 25S rRNA have been elucidated, as well as the primary structures of high-molecular-weight (28S) rRNAs of Eukaryotic cells, which number about 4700 nucleotides.

Currently, research into the primary structures of various DNA molecules is actively underway. About 15 years ago, the nucleotide sequence of human Mitochondrial DNA (16,569 Base Pairs) was completely deciphered. The complete nucleotide sequences of DNA from a number of Viruses and Plasmids are also known. Very recently, the Determination of the nucleotide sequences of the genomes of two prokaryotic organisms (Haemophilus influenzae and Mycoplasma genitalium) was completed, and reports emerged regarding the deciphering of The Genome of the first eukaryotic Organism—yeast. Similar studies on the E. coli genome and the Caenorhabditis elegans nematode genome are nearing completion. Researchers are actively working on fully sequencing The Human Genome.

The results of sequencing (determining the nucleotide sequence) of various DNA molecules are accumulated in computer Databases that are already accessible to users of international computer networks (such as the Internet). Three Variants of the DNA nucleotide sequence diagram are presented below:

Recently, the primary structure of DNA (or more precisely, its individual fragments) has been inferred from a number of indirect data, such as the degree of clustering of nucleotide units in the DNA molecule (this analysis ultimately boils down to determining the number and structure of individual nucleotide fractions, the so-called isopliths), as well as from DNA reassociation kinetics (a method that reveals the presence of repeating nucleotide sequences within the molecule). The primary structure of DNA is also evaluated based on the distribution of minor bases (evidence suggests the existence of such a regularity) and the detection and Sequence Determination of palindromes ("inverted repeats"), which are found primarily at restriction sites (see Chapter 13). Researchers place high hopes on physical, chemical (gene synthesis), genetic, and other Methods for determining DNA primary structure, as well as on methods for isolating specific genes (or their fragments) from natural sources and synthesizing genes from mRNA using the enzyme Reverse Transcriptase. To establish the primary structure of DNA, a rapid method has recently been proposed involving The Use of two DNA polymerases (from E. coli and bacteriophage T4). However, in all these cases, only The structure of a small DNA region is determined, and therefore the complete deciphering of the primary structure of the human genome remains a challenge for the future.