BIOCHEMISTRY - L. Stryer - 1984

VOLUME 3

Part IV INFORMATION

CHAPTER 25. RNA INFORMATION AND TRANSCRIPTION

25.18. Antibiotics as Transcription Inhibitors: Rifamycin and Actinomycin

Antibiotics are fascinating molecules, as many of them serve as highly specific inhibitors of biological processes. Actinomycin and rifamycin are two antibiotics that inhibit METABOLISM/31.html">Transcription through entirely different mechanisms. Rifamycin, produced by Streptomyces Bacteria, and its semisynthetic derivative rifampicin specifically inhibit the initiation of RNA Synthesis. These antibiotics do not prevent the binding of RNA polymerase to the DNA template. Rifampicin blocks The formation of the first phosphodiester bond in the RNA chain, while having virtually no effect on chain elongation. This high selectivity of inhibitory action makes rifampicin a valuable tool in molecular biology research. For instance, it can be used to suppress the initiation of new RNA chains without affecting the transcription of chains whose synthesis has already begun. The target for rifampicin appears to be the β-subunit of RNA polymerase. Rifampicin-resistant E. coli mutants (so-called rif-r mutants) have been isolated, and some of them exhibit an altered electrophoretic mobility of the β-subunit.

Class="center">Fig. 25.21. Spatial model of the actinomycin D Structure. The phenoxazone ring is shown in red, and the cyclic Peptides are shown in yellow.

The MECHANISM OF ACTION of actinomycin D (a polypeptide-containing antibiotic produced by the microorganism Streptomyces) is entirely different from that of rifampicin. Actinomycin D binds tightly to double-helical DNA, thereby inhibiting its activity as a template for RNA synthesis. It consists of two identical cyclic peptides linked by a phenoxazone ring system (Fig. 25.22). The composition of these cyclic peptides is unusual: they contain Sarcosine, methylvaline, and D-valine. Furthermore, their molecules contain an ester bond between the hydroxyl group of Threonine and the carboxyl group of methylvaline.

Fig. 25.22. Structure of actinomycin D

Actinomycin D binds tightly to double-helical DNA, but not to single-stranded DNA, RNA, double-stranded RNA, or RNA-DNA hybrids. Moreover, the binding of actinomycin to DNA is markedly enhanced as the guanine residue content increases. Spectroscopic and hydrodynamic studies of actinomycin D-DNA complexes indicate that the phenoxazone ring of actinomycin intercalates into DNA between two adjacent Base Pairs. This mode of binding is known as intercalation (Fig. 25.23). At low concentrations, actinomycin D inhibits transcription without exerting any significant effect on DNA Replication. Protein Synthesis is also not directly affected by actinomycin. Consequently, actinomycin D has been widely used as a specific inhibitor of new RNA synthesis in both prokaryotic and Eukaryotic Cells.

Fig. 25.23. Proposed STRUCTURE OF THE actinomycin D-DNA complex. The phenoxazone ring of actinomycin (shown in red) intercalates between two GC base pairs of DNA (shown in blue). The cyclic peptides of actinomycin D (yellow) bind to the minor groove of the DNA helix. Several Hydrogen Bonds are formed between actinomycin D and adjacent guanines. The Symmetry axis of the actinomycin D subunits coincides with the symmetry axis of the sugar-phosphate backbone and the DNA base sequence.

Recently, the crystal structure of a complex between one actinomycin molecule and two deoxyguanosine molecules was investigated at atomic resolution (Fig. 25.23). In this complex, the phenoxazone ring of actinomycin is sandwiched between two guanine rings. One of the cyclic peptides is positioned above the phenoxazone ring, and the other below it. Each of these peptides forms strong hydrogen bonds with the 2-amino group of the guanine residue. Numerous energetically favorable Van der Waals interactions occur between the antibiotic and the nucleosides. An important feature of this complex is that it is nearly symmetrical. A twofold axis of symmetry runs along the line connecting the middle O and N atoms of the phenoxazone ring. The conformation of the entire complex demonstrates that actinomycin recognizes the GpC base sequence in DNA. Note that if a 5'-GpC-3' sequence is present in One DNA strand, the complementary strand will have the 3'-CpG-5' sequence. Apparently, actinomycin intercalates into DNA between two GC base pairs and interacts with the G residues in much the same way as in the complex with the dinucleotide. According to this model, the cyclic peptide residues are located within the minor groove of the DNA helix. The primary feature of this model is that the symmetry of the actinomycin molecule matches the symmetry of a specific sequence in DNA.

25.19. Advanced Methods for Determining Nucleotide Sequences in RNA Have Been Developed

The high resolution of gel Electrophoresis makes it possible to rapidly determine nucleotide sequences in both RNA molecules and

DNA molecules. To achieve this, the 3'- or 5'-end of the RNA chain is labeled with a radioactive group. The labeled chain is then partially cleaved at one of the four bases to generate a set of fragments. Specific (or selective) Cleavage can be achieved using base-specific Enzymes known as endonucleases (Table 25.4). For example, Ribonuclease T₁ hydrolyzes RNA on the 3' side of G residues. Alternatively, RNA can be specifically cleaved via chemical modification of one of the four bases, followed by Cleavage of the backbone at the site of the modified base. The four sets of fragments are then separated by gel electrophoresis, and the RNA base sequence is read directly from the autoradiogram (Fig. 25.24), similarly to DNA Sequencing (Section 24.28). These approaches make it straightforward to determine sequences of 100 to 200 NUCLEOTIDES in RNA molecules.

Fig. 25.24. Autoradiogram of a Yeast 5S RNA fragment. The 3'-end carries a radioactive label. The four lanes correspond to cleavage at G, A, C, and U residues, respectively. The sequence 5'-CGAAACUCAGGUGCUGCAAUC-3' can be read from the gel.

Table 25.4. Enzymes used in determining nucleotide sequences in RNA¹

¹ The presented viewpoint on The structure of the actinomycin D-DNA complex is not universally accepted. There is an alternative hypothesis suggesting that actinomycin does not intercalate into DNA but rather binds in the minor groove, and that the structure of the actinomycin D-dinucleotide GpC complex does not reflect the structure of its complexes with extended DNA molecules. — Translator's Note.

Another strategy is to determine The nucleotide sequence not in the RNA itself, but in the complementary DNA. But how can complementary DNA fragments be obtained? One approach involves fragmenting large DNA molecules using Restriction Endonucleases. Restriction fragments containing the sequence complementary to the RNA of interest are then identified by Hybridization. Alternatively, complementary DNA can be synthesized enzymatically from an RNA template using Reverse Transcriptase from Oncogenic Viruses (Section 30.19). This is precisely how the complete sequence of the 576 nucleotides of the mRNA encoding the human Hemoglobin β-chain was deciphered.