BIOCHEMISTRY - L. Stryer - 1984

VOLUME 3

Part IV INFORMATION

CHAPTER 25. RNA INFORMATION AND TRANSCRIPTION

25.5. Hybridization Experiments Reveal that Messenger RNA is Complementary to its DNA Template

In 1961, Sol Spiegelman developed a novel technique known as Hybridization. It was designed to answer a fundamental question: Is The base sequence of RNA synthesized following phage T2 infection complementary to the base sequence of phage T2 DNA? Julius Marmur and Paul Doty had previously shown that when double-helical DNA is heated above its melting Temperature, it converts into a single-stranded form. Upon slow cooling of the solution, these strands reassociate to form a biologically active double-helical Structure. Marmur and Doty also discovered that double-helical molecules form only if the DNA strands originate from the same species or closely related species. This observation suggested to Spiegelman that DNA-RNA hybrids should form in a mixture of single-stranded DNA and RNA provided their base sequences are complementary (Fig. 25.5). The experimental design was as follows:

1. RNA synthesized following E. coli infection with phage T2 (T2 mRNA) was labeled with the ³²P isotope. In a separate experiment, phage T2 DNA (T2 DNA) was labeled with the ³H isotope.

2. A mixture of T2 mRNA and T2 DNA was heated to 100°C. As a result, the double-helical DNA melted and converted into single-stranded form. This solution, containing single-stranded RNA and DNA, was slowly cooled to room temperature.

3. The cooled mixture was analyzed by density gradient centrifugation. The samples were centrifuged for several days in a swinging-bucket rotor. Afterward, the plastic centrifuge tubes were punctured from the bottom, and drop fractions were collected for further analysis.

Class="center">Fig. 25.5. If RNA and DΝΑ have complementary sequences, an RNA-DNA hybrid can form

As a result, three distinct bands were detected (Fig. 25.6). The band with the highest density corresponded to single-stranded RNA. The second band corresponded to double-helical DNA. The third was located close to the DNA band and consisted of double-stranded DNA-RNA hybrid molecules. Thus, T2 mRNA formed a hybrid with T2 DNA. In contrast, T2 RNA did not hybridize with the DNA of numerous Bacteria and unrelated Viruses, even when their nucleotide composition was similar to that of T2 DNA. Subsequent experiments demonstrated that the mRNA fraction from uninfected Cells hybridizes specifically with the DNA of the Organism from which it was isolated, but not with the DNA of unrelated organisms. These compelling experiments proved that the base sequence of mRNA is complementary to the template DNA sequence. Furthermore, they provided a powerful method for investigating the flow of Genetic information within cells and determining whether two nucleic acid molecules are related.

Fig. 25.6. RNA synthesized following E. coli infection with phage T2 is complementary to viral DNA. In these hybridization experiments, RNA was labeled with ³²P and phage T2 DNA with ³H. The distribution of radioactivity in a cesium chloride density gradient shows that the majority of RNA synthesized post-infection cosediments in a single band with phage T2 DNA

25.6. Ribosomal RNAs and Transfer RNAs Are Also Synthesized on a DNA Template

The hybridization technique was subsequently employed to determine whether rRNA and tRNA are also synthesized on DNA templates. The formation of RNA-DNA hybrids was detected using membrane filters rather than density gradient centrifugation, as this approach is simpler, more sensitive, and less time-consuming. Single-stranded RNAs pass through a nitrocellulose filter, whereas double-helical DNAs and RNA-DNA hybrids are retained on the filter. E. coli RNA was labeled with ³²P and mixed with unlabeled E. coli DNA. This mixture was heated, slowly cooled, and then filtered through nitrocellulose. The radioactivity retained on the filter was quantified. The experimental results were unambiguous: RNA-DNA hybrids formed with all rRNAs (5S, 16S, and 23S) and tRNAs. This indicated that the E. coli genome contains sequences complementary to these RNA molecules.

25.7. All Cellular RNAs Are Synthesized by RNA Polymerase

METABOLISM/2.html">THE CONCEPT OF mRNA spurred the search for an enzyme capable of synthesizing RNA directed by a DNA template. The experimental strategy mirrored the search for DNA polymerase I. In 1960, Jerard Hurwitz and Samuel Weiss independently discovered such an enzyme, which they named RNA polymerase. The enzyme isolated from E. coli cells (Fig. 25.7) required the following components for RNA Synthesis.

Fig. 25.7. Electron micrograph of E. coli RNA polymerase

1. Template. The preferred template is double-stranded DNA. Single-stranded DNA can also serve as a template. Neither RNA (single- nor double-stranded) nor RNA-DNA hybrids can function as effective templates.

2. Activated precursors. All four ribonucleoside triphosphates—ATP, GTP, UTP, and CTP—are required.

3. Divalent Metal Ions. The enzyme is active in the presence of Mg²⁺ or Mn²⁺. In vivo, this enzymatic requirement is fulfilled by Mg²⁺.

RNA polymerase catalyzes the initiation and elongation of RNA chains. The enzyme catalyzes the following reaction:

RNA synthesis is in many respects similar to DNA Synthesis (Fig. 25.8). First, as will soon be discussed in greater detail, synthesis proceeds in the 5' → 3' direction. Second, the elongation mechanism appears to be comparable. It involves a nucleophilic attack of the 3'-OH group at the end of the growing chain on the inner phosphate of an incoming nucleoside triphosphate. Third, the driving force of synthesis is the Hydrolysis of pyrophosphate.

Fig. 25.8. Mechanism of the chain elongation reaction catalyzed by RNA polymerase

However, RNA synthesis differs from DNA synthesis in several important features. First, RNA polymerase does not require a primer. Second, the DNA template is entirely conserved during RNA synthesis, whereas in DNA synthesis it is only half conserved. Third, RNA polymerase is not known to exhibit any nuclease activities.

All Three types of cellular RNA in E. coli — mRNA, tRNA, and rRNA — are synthesized by a single RNA polymerase According to the instructions provided by the DNA template. In mammalian cells, There is a division of labor among several types of RNA polymerases. In addition, it should be noted that some viruses encode RNA-synthesizing Enzymes that are completely distinct from those of the host Cell. Examples include the RNA polymerase encoded by the DNA-containing phage T7 and the RNA replicase encoded by the RNA-containing phage Qß. Qß phage replicase is an RNA-dependent RNA polymerase, as it uses RNA rather than a DNA template to synthesize RNA (Ch. 30). In contrast, cellular enzymes that synthesize RNA are DNA-dependent RNA polymerases.