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Berg JM, Tymoczko JL, Stryer L. Biochemistry. 5th edition. New York: W H Freeman; 2002.

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Biochemistry. 5th edition.

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Section 5.4 Gene Expression Is the Transformation of DNA Information Into Functional Molecules

The information stored as DNA becomes useful when it is expressed in the production of RNA and proteins. This rich and complex topic is the subject of several chapters later in this book, but here we introduce the basics of gene expression. DNA can be thought of as archival information, stored and manipulated judiciously to minimize damage (mutations). It is expressed in two steps. First, an RNA copy is made. An RNA molecule that encodes proteins can be thought of as a photocopy of the original information—it can be made in multiple copies, used, and then disposed of. Second, an RNA molecule can be further thought of as encoding directions for protein synthesis that must be translated to be of use. The information in messenger RNA is translated into a functional protein. Other types of RNA molecules exist to facilitate this translation. We now examine the transcription of DNA information into RNA, the translation of RNA information into protein, and the genetic code that links nucleotide sequence with amino acid sequence.

5.4.1. Several Kinds of RNA Play Key Roles in Gene Expression

Cells contain several kinds of RNA (Table 5.2).

Table 5.2. RNA molecules in E. coli.

Table 5.2

RNA molecules in E. coli.

1.

Messenger RNA is the template for protein synthesis or translation. An mRNA molecule may be produced for each gene or group of genes that is to be expressed in E. coli, whereas a distinct mRNA is produced for each gene in eukaryotes. Consequently, mRNA is a heterogeneous class of molecules. In E. coli, the average length of an mRNA molecule is about 1.2 kilobases (kb).

2.

Transfer RNA carries amino acids in an activated form to the ribosome for peptide-bond formation, in a sequence dictated by the mRNA template. There is at least one kind of tRNA for each of the 20 amino acids. Transfer RNA consists of about 75 nucleotides (having a mass of about 25 kd), which makes it the smallest of the RNA molecules.

3.

Ribosomal RNA (rRNA) ,the major component of ribosomes, plays both a catalytic and a structural role in protein synthesis (Section 29.3.1). In E. coli, there are three kinds of rRNA, called 23S, 16S, and 5S RNA because of their sedimentation behavior. One molecule of each of these species of rRNA is present in each ribosome.

Kilobase (kb)—

A unit of length equal to 1000 base pairs of a double-stranded nucleic acid molecule (or 1000 bases of a single-stranded molecule).

One kilobase of double-stranded DNA has a contour length of 0.34 μm and a mass of about 660 kd.

Ribosomal RNA is the most abundant of the three types of RNA. Transfer RNA comes next, followed by messenger RNA, which constitutes only 5% of the total RNA. Eukaryotic cells contain additional small RNA molecules. Small nuclear RNA (snRNA) molecules, for example, participate in the splicing of RNA exons. A small RNA molecule in the cytosol plays a role in the targeting of newly synthesized proteins to intracellular compartments and extracellular destinations.

5.4.2. All Cellular RNA Is Synthesized by RNA Polymerases

The synthesis of RNA from a DNA template is called transcription and is catalyzed by the enzyme RNA polymerase (Figure 5.24). RNA polymerase requires the following components:

Figure 5.24. RNA Polymerase.

Figure 5.24

RNA Polymerase. Image mouse.jpg A large enzyme comprising many subunits including β (red) and β′ (blue), which form a “claw” that holds the DNA to be transcribed. The active site includes a Mg2+ ion at the center of the structure. (more...)

1.

A template. The preferred template is double-stranded DNA. Single-stranded DNA also can serve as a template. RNA, whether single or double stranded, is not an effective template; nor are RNA-DNA hybrids.

2.

Activated precursors. All four ribonucleoside triphosphatesATP, GTP, UTP, and CTP—are required.

3.

A divalent metal ion. Mg2+ or Mn2+ are effective.

RNA polymerase catalyzes the initiation and elongation of RNA chains. The reaction catalyzed by this enzyme is:

Image ch5e3.jpg

The synthesis of RNA is like that of DNA in several respects (Figure 5.25). First, the direction of synthesis is 5′ → 3′. Second, the mechanism of elongation is similar: the 3′-OH group at the terminus of the growing chain makes a nucleophilic attack on the innermost phosphate of the incoming nucleoside triphosphate. Third, the synthesis is driven forward by the hydrolysis of pyrophosphate. In contrast with DNA polymerase, however, RNA polymerase does not require a primer. In addition, RNA polymerase lacks the nuclease capability used by DNA polymerase to excise mismatched nucleotides.

Figure 5.25. Transcription Mechanism of the Chain-Elongation Reaction Catalyzed by RNA Polymerase.

Figure 5.25

Transcription Mechanism of the Chain-Elongation Reaction Catalyzed by RNA Polymerase.

All three types of cellular RNAmRNA, tRNA, and rRNA—are synthesized in E. coli by the same RNA polymerase according to instructions given by a DNA template. In mammalian cells, there is a division of labor among several different kinds of RNA polymerases. We shall return to these RNA polymerases in Chapter 28.

5.4.3. RNA Polymerases Take Instructions from DNA Templates

RNA polymerase, like the DNA polymerases described earlier, takes instructions from a DNA template. The earliest evidence was the finding that the base composition of newly synthesized RNA is the complement of that of the DNA template strand, as exemplified by the RNA synthesized from a template of single-stranded φX174 DNA (Table 5.3). Hybridization experiments also revealed that RNA synthesized by RNA polymerase is complementary to its DNA template. In these experiments, DNA is melted and allowed to reassociate in the presence of mRNA. RNA-DNA hybrids will form if the RNA and DNA have complementary sequences. The strongest evidence for the fidelity of transcription came from base-sequence studies showing that the RNA sequence is the precise complement of the DNA template sequence (Figure 5.26).

Table 5.3. Base composition (percentage) of RNA synthesized from a viral DNA template.

Table 5.3

Base composition (percentage) of RNA synthesized from a viral DNA template.

Figure 5.26. Complementarity between mRNA and DNA.

Figure 5.26

Complementarity between mRNA and DNA. The base sequence of mRNA (red) is the complement of that of the DNA template strand (blue). The sequence shown here is from the tryptophan operon, a segment of DNA containing the genes for five enzymes that catalyze (more...)

5.4.4. Transcription Begins near Promoter Sites and Ends at Terminator Sites

RNA polymerase must detect and transcribe discrete genes from within large stretches of DNA. What marks the beginning of a transcriptional unit? DNA templates contain regions called promoter sites that specifically bind RNA polymerase and determine where transcription begins. In bacteria, two sequences on the 5′ (upstream) side of the first nucleotide to be transcribed function as promoter sites (Figure 5.27A). One of them, called the Pribnow box, has the consensus sequence TATAAT and is centered at -10 (10 nucleotides on the 5′ side of the first nucleotide transcribed, which is denoted by + 1). The other, called the -35 region, has the consensus sequence TTGACA. The first nucleotide transcribed is usually a purine.

Consensus sequence—

The base sequences of promoter sites are not all identical. However, they do possess common features, which can be represented by an idealized consensus sequence. Each base in the consensus sequence TATAAT is found in a majority of prokaryotic promoters. Nearly all promoter sequences differ from this consensus sequence at only one or two bases.

Figure 5.27. Promoter Sites for Transcription.

Figure 5.27

Promoter Sites for Transcription. Promoter sites are required for the initiation of transcription in both (A) prokaryotes and (B) eukaryotes. Consensus sequences are shown. The first nucleotide to be transcribed is numbered +1. The adjacent nucleotide (more...)

Eukaryotic genes encoding proteins have promoter sites with a TATAAA consensus sequence, called a TATA box or a Hogness box, centered at about -25 (Figure 5.27B). Many eukaryotic promoters also have a CAAT box with a GGNCAATCT consensus sequence centered at about -75. Transcription of eukaryotic genes is further stimulated by enhancer sequences, which can be quite distant (as many as several kilobases) from the start site, on either its 5′ or its 3′ side.

RNA polymerase proceeds along the DNA template, transcribing one of its strands until it reaches a terminator sequence. This sequence encodes a termination signal, which in E. coli is a base-paired hairpin on the newly synthesized RNA molecule (Figure 5.28). This hairpin is formed by base pairing of self-complementary sequences that are rich in G and C. Nascent RNA spontaneously dissociates from RNA polymerase when this hairpin is followed by a string of U residues. Alternatively, RNA synthesis can be terminated by the action of rho, a protein. Less is known about the termination of transcription in eukaryotes. A more detailed discussion of the initiation and termination of transcription will be given in Chapter 28. The important point now is that discrete start and stop signals for transcription are encoded in the DNA template.

Figure 5.28. Base Sequence of the 3′ end of an mRNA transcript in E. coli.

Figure 5.28

Base Sequence of the 3′ end of an mRNA transcript in E. coli. A stable hairpin structure is followed by a sequence of uridine (U) residues.

In eukaryotes, the mRNA is modified after transcription (Figure 5.29). A “cap” structure is attached to the 5′ end, and a sequence of adenylates the poly(A) tail is added to the 3′ end. These modifications will be presented in detail in Section 28.3.1.

Figure 5.29. Modification of mRNA.

Figure 5.29

Modification of mRNA. Messenger RNA in eukaryotes is modified after transcription. A nucleotide “cap” structure is added to the 5′ end, and a poly(A) tail is added at the 3′ end.

5.4.5. Transfer RNA Is the Adaptor Molecule in Protein Synthesis

We have seen that mRNA is the template for protein synthesis. How then does it direct amino acids to become joined in the correct sequence to form a protein? In 1958, Francis Crick wrote:

RNA presents mainly a sequence of sites where hydrogen bonding could occur. One would expect, therefore, that whatever went onto the template in a specific way did so by forming hydrogen bonds. It is therefore a natural hypothesis that the amino acid is carried to the template by an adaptor molecule, and that the adaptor is the part that actually fits onto the RNA. In its simplest form, one would require twenty adaptors, one for each amino acid.

This highly innovative hypothesis soon became established as fact. The adaptor in protein synthesis is transfer RNA. The structure and reactions of these remarkable molecules will be considered in detail in Chapter 29. For the moment, it suffices to note that tRNA contains an amino acidattachment site and a template-recognition site. A tRNA molecule carries a specific amino acid in an activated form to the site of protein synthesis. The carboxyl group of this amino acid is esterified to the 3′- or 2′-hydroxyl group of the ribose unit at the 3′ end of the tRNA chain (Figure 5.30). The joining of an amino acid to a tRNA molecule to form an aminoacyl-tRNA is catalyzed by a specific enzyme called an aminoacyl-tRNA synthetase (or acti-vating enzyme). This esterification reaction is driven by ATP. There is at least one specific synthetase for each of the 20 amino acids. The template-recognition site on tRNA is a sequence of three bases called an anticodon (Figure 5.31). The anticodon on tRNA recognizes a complementary sequence of three bases, called a codon, on mRNA.

Figure 5.30. Attachment of an Amino Acid to a tRNA Molecule.

Figure 5.30

Attachment of an Amino Acid to a tRNA Molecule. The amino acid (shown in blue) is esterified to the 3′-hydroxyl group of the terminal adenosine of tRNA.

Figure 5.31. Symbolic Diagram of an Aminoacyl-tRNA.

Figure 5.31

Symbolic Diagram of an Aminoacyl-tRNA. The amino acid is attached at the 3′ end of the RNA. The anticodon is the template-recognition site.

By agreement with the publisher, this book is accessible by the search feature, but cannot be browsed.

Copyright © 2002, W. H. Freeman and Company.
Bookshelf ID: NBK22518

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