BIOCHEMISTRY - L. Stryer - 1984

VOLUME 3

Part IV INFORMATION

CHAPTER 25. RNA INFORMATION AND TRANSCRIPTION

25.8. RNA Polymerase Receives Instructions from the DNA Template

Like the DNA polymerases described in the previous chapter, RNA polymerase utilizes the information contained within a DNA template. The first evidence supporting this notion came from findings showing that the Nucleotide Composition of newly synthesized RNA is complementary to that of the template DNA strand. When the synthetic polydeoxyribonucleotide poly(dT)—which contains only thymidylate residues—is used as a template, only a single ribonucleoside triphosphate, ATP, is incorporated into the growing polyribonucleotide chain. The reaction product is polyriboadenylate [abbreviated as poly(rA)]. If the alternating copolymer poly(dA-dT) is used as a template for RNA polymerase, both UTP and ATP are incorporated, yielding poly(rA-rU) as the reaction product. The nucleotide composition of RNA synthesized using single-stranded DNA from the ɸX174 phage as a template further demonstrates complementarity between the RNA product and the DNA template (Table 25.2). Hybridization experiments reveal that the RNA synthesized by RNA polymerase

is complementary to the template DNA. The most compelling proof for The fidelity of DNA copying during METABOLISM/31.html">Transcription comes from sequence analysis; it turns out that the mRNA sequence is precisely complementary to the template DNA sequence (Fig. 25.9).

Class="center">Fig. 25.9. The base sequence of mRNA is complementary to that of the template DNA. A portion of the Tryptophan Operon sequence is shown

Table 25.2. Nucleotide composition of RNA synthesized on a viral DNA template

25.9. Typically, Only One DNA Strand Is Transcribed in a Given Region of the Genome

Are both strands of a double-helical DNA template transcribed, or only one? A priori, it seems unlikely that both strands within the same region of DNA would encode functional Proteins. One of the first Answers to this question came from hybridization experiments using E. coli infected with phage ɸX174. Phage ɸX174 particles contain a single-stranded DNA known as the plus strand. Once the plus strand enters the bacterium, a complementary minus strand is synthesized, yielding a circular double-stranded DNA molecule called the replicative form (RF). This RF-DNA directs the synthesis of mRNA, which in turn determines the phage proteins. Shortly after E. coli was infected with phage ɸX174, ³²P-phosphate was added to the medium to radiabel the phage RNA, which was subsequently isolated. In addition, the RF-DNA was separated into its constituent plus and minus strands—a straightforward task since they differ in nucleotide composition and, consequently, in buoyant density. Hybridization was then performed to determine whether the newly synthesized mRNA was complementary to the plus strand, the minus strand, or both. An unequivocal result was obtained: only the minus strand of the RF-DNA formed a hybrid with the labeled mRNA. Thus, only one strand of phage ɸX174 RF-DNA serves as the template for transcription in vivo.

Fig. 25.10. As this hybridization experiment demonstrates, only one strand of phage ɸX174 RF-DNA is used as a template during transcription

Fig. 25.11. An electron micrograph showing The process of transcription

Similarly, only one strand of Viruses such as T7, SP8, and α is transcribed in vivo. For viruses such as phages T4 or , the situation is more complex. In some Regions of the genome, one strand serves as the template, while in others, the opposite strand does. Such strand-switching transcription across different Gene groups also occurs in E. coli Cells.

25.10. E. coli RNA Polymerase Consists of Subunits

E. coli RNA polymerase is a very large and complex enzyme, with a molecular mass of the holoenzyme of approximately 500 kDa. RNA polymerase is composed of distinct subunits (Table 25.3), as can be demonstrated by inducing dissociation of the enzyme in a concentrated urea solution. The subunit COMPOSITION OF THE complete enzyme, known as the holoenzyme, is α₂ββ'σ. As will be seen below, once the initiation of RNA Synthesis has occurred, the σ subunit dissociates from the enzyme. RNA polymerase lacking the σ subunit is called the core enzyme (α₂ββ'). The catalytic site of RNA polymerase resides within the core enzyme. It has been established that the β' subunit participates in binding to the DNA template, whereas the β subunit is involved in binding the substrates, ribonucleoside triphosphates. The σ subunit of the holoenzyme participates in selecting the Transcription initiation site.

Table 25.3. Subunits of E. coli RNA Polymerase

Fig. 25.12. An electron micrograph of RNA polymerase holoenzyme bound to multiple promoter regions of a T7 phage DNA fragment

RNA synthesis by E. coli RNA polymerase proceeds in three stages, termed 1) initiation, 2) elongation, and 3) termination. We will examine these processes shortly.