Introduction to Molecular Biology: From Cells to Atoms - Anthony Rees, Michael Sternberg 2002

Nucleic Acids and Genes
RNA Structure

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Fig. 19.1.

Ribonucleic Acids (RNAs), found in both prokaryotic and Eukaryotic Cells, occur in three main types: Messenger RNA (mRNA), Transfer RNA (tRNA), and ribosomal RNA (rRNA).

Eukaryotic Cell nuclei contain a fourth type of RNA known as heterogeneous nuclear RNA (hnRNA). In some Viruses, RNA serves as the carrier of Genetic information (Ch. 5). mRNA is a copy (transcript) of the corresponding DNA, serving as a template for Protein Synthesis. Every three consecutive bases in mRNA (referred to as a codon) specify a single amino acid residue. tRNA molecules deliver specific amino acid residues to the appropriate site on the mRNA during protein Translation. The Structure of tRNA is currently well understood (see below). rRNA molecules exist in various forms and combine with Proteins to form Ribosomes (Ch. 24), the complex Organelles where protein synthesis takes place.

The Svedberg (S) unit measures the sedimentation coefficient, which serves as an indicator of macromolecular mass (Ch. 44). It is determined by measuring the rate at which molecules settle in a centrifugal field. The sedimentation coefficients for E. coli RNA are listed in Table 19.1.

Transfer RNA (tRNA) recognizes the corresponding codon on the mRNA and delivers the required amino acid to the growing polypeptide chain. Codon recognition on the mRNA is mediated by three consecutive bases in the tRNA, known as the anticodon. The amino acid residue attaches to the 3'-end of the tRNA molecule. The Specificity of this transport system is ensured by the presence of at least one specific tRNA for each amino acid. For instance, the tRNA specific for phenylalanine is denoted as tRNA_Phe.

Table 19.1. Characteristics of various E. coli RNAs

Chemical Structure of tRNA. A tRNA molecule consists of roughly 75 NUCLEOTIDES covalently linked into a linear chain, with a molecular mass M_r = 25,000 and a sedimentation coefficient of 4S. The nucleotide sequences of numerous tRNAs have been mapped; the first of these—Yeast tRNA_Ala—was sequenced by Holley and coworkers in 1965. All tRNAs begin with a phosphorylated 5'-end, where the first base is typically G. The 3'-end invariably features a CCA triplet followed by a terminal 3'-hydroxyl (—OH) group. tRNA composition includes not only the four standard bases (A, C, G, and U), but also several modified, or minor, bases. For example, yeast tRNA_Phe contains pseudouridine (denoted as Ψ), dihydrouridine (D), and a characteristic base designated as Y. Furthermore, one or two hydrogen atoms in certain standard bases may be replaced by methyl (CH₃) groups; these methylated or dimethylated bases are indicated by the symbols m or m₂ prefixed to the respective base letter.

The cloverleaf model is a two-dimensional structural representation of a tRNA molecule, derived by maximizing the number of Watson-Crick Base Pairs possible for a given nucleotide sequence. The regions formed by these hydrogen-bonded base pairs are called stems, whereas the unpaired stretches are known as loops. All known tRNAs fold into a "cloverleaf" comprising four stems (the acceptor, D, anticodon, and TΨC stems) and three corresponding loops (the D, anticodon, and TΨC loops). Certain tRNAs possess extra loops and/or stems (such as the variable loop in yeast tRNA_Phe).

The three-dimensional structure of yeast tRNA_Phe was elucidated using X-ray crystallography. While the term "Secondary structure" refers to the spatial arrangement of atoms within the stems, the tertiary structure describes the overall three-dimensional conformation of the entire molecule. Secondary Structure: Each stem is composed of two antiparallel strands whose bases are linked by Watson-Crick Hydrogen Bonds. This spatial arrangement, confirmed experimentally, was accurately predicted from the cloverleaf model. The stems adopt the conformation of a right-handed double helix, known as the A-form of RNA.

The A-form RNA double helix contains 11 base pairs per turn, with a pitch of 3.1 nm. The distance between adjacent base pairs along the helical axis is 0.28 nm (3.1 nm/11), and they are rotated relative to one another by an angle of 33° (360°/11). The planes of the base pairs are tilted at an angle of ~20° relative to the normal to the helical axis. The A-form of RNA closely resembles the A-form of DNA (Ch. 18). RNA is unable to transition into the B-form due to the steric hindrance of the bulky 2'-hydroxyl group on the ribose ring, which is absent in deoxyribose.

Tertiary Structure of tRNA. The molecule adopts an overall L-shaped conformation, making it significantly more elongated than Globular proteins of similar molecular mass. The acceptor and T-stems are stacked in space to form one continuous helix—the "crossbar" of the L—while the anticodon and D-stems form the "upright". Each of these two domains contains approximately 10 base pairs. Nearly all bases in tRNA participate in Van der Waals and hydrophobic interactions that stabilize the spatial architecture of the molecule.

A wide variety of Hydrogen bonds are present in tRNA. Watson-Crick hydrogen bonds stabilize the double-helical stems. In addition, many bases located outside the stems form non-Watson-Crick hydrogen bonds with other bases. Furthermore, the 2'-hydroxyl groups of the polynucleotide backbone also form hydrogen bonds with nucleotide bases and backbone atoms.

The anticodon of a tRNA molecule consists of three consecutive bases responsible for recognizing the complementary codon on the mRNA. This recognition occurs via The formation of Watson-Crick hydrogen bonds between the codon bases and the anticodon bases, provided that the polynucleotide strands are antiparallel (Fig. 19.2).

Fig. 19.2.

OTHER RNA MOLECULES. These include mRNA and single-stranded viral RNA, whose nucleotide sequences have also been determined experimentally. These studies demonstrate that specific regions of RNA molecules can form hydrogen-bonded hairpin loops, much like tRNA. Double-stranded viral RNA appears to adopt an A-like conformation. Further high-resolution X-ray crystallographic studies are required to fully elucidate the structural details of all these molecules.

RNA SEQUENCING can now be performed rapidly, yielding sequences of several hundred bases at a time. Previously, this Procedure was exceptionally tedious and time-consuming, limiting researchers to RNA fragments of no more than 100 bases (as in Holley's 1965 work; see above). Today, a variety of Methods are available, generally following two main approaches: the direct approach and the copying method.

The direct approach involves sequencing the RNA molecule directly. First, the 5'- or 3'-end of the RNA is radiolabeled using ³²PO₄. The labeled chain is then cleaved using sequence-specific Enzymes. By performing gel Electrophoresis and autoradiography, a ladder is generated that reveals The base sequence, much like the sequencing of DNA (Ch. 18).

In the copying method, a complementary DNA (cDNA) strand is first synthesized from the RNA template, and its nucleotide sequence is then determined using the techniques described in Chapter 18. This complementary DNA strand is typically synthesized using Reverse Transcriptase, an enzyme discovered in certain viruses that carries out reverse METABOLISM/31.html">Transcription—catalyzing the synthesis of a DNA strand (known as cDNA) complementary to an RNA template. (Reverse transcription also plays a pivotal role in Gene cloning; see Ch. 30.) For instance, if an RNA segment has the sequence 3'-GGGUCA, the resulting cDNA will have the sequence 5'-GCCAGT. Applying chemical DNA Sequencing Methods (Ch. 18) to this cDNA produces an autoradiogram such as the one shown in Fig. 19.3.

Fig. 19.3.