The simplest definition of a gene is “a unit of DNA that contains the information to specify synthesis of a single poly peptide chain or functional RNA (such as a tRNA).” The DNA molecules of small viruses contain only a few genes, whereas the single DNA molecule in each of the chromosomes of higher animals and plants may contain several thousand genes. The vast majority of genes carry information used to build protein molecules, and it is the RNA copies of such protein-coding genes that constitute the mRNA molecules of cells.

During synthesis of RNA, the four-base language of DNA containing A, G, C, and T is simply copied, or transcribed, into the four-base language of RNA, which is identical except that U replaces T. In contrast, during protein synthesis, the four-base language of DNA and RNA is translated into the 20–amino acid language of proteins. In this section, we focus on the formation of functional mRNAs from protein coding genes. A similar process yields precursors of rRNAs and tRNAs, encoded by rRNA and tRNA genes; these precursors are then further modified to yield functional rRNAs and tRNAs. Similarly, thousands of micro-RNAs (miRNAs), which regulate the translation and stability of specific target mRNAs, are transcribed into precursors by RNA polymerases and processed into functional miRNAs. Other non-protein-coding (or simply noncoding) RNAs help to regulate the transcription of specific protein-coding genes. Regulation of transcription allows distinct sets of genes to be expressed in the multiple different types of cells that make up a multicellular organism. It also allows different amounts of mRNA to be transcribed from different genes, resulting in differences in the amounts of the encoded proteins in a cell.

A Template DNA Strand Is Transcribed into a Complementary RNA Chain by RNA Polymerase

 During transcription of DNA, one DNA strand acts as a template, determining the order in which ribonucleoside triphosphate (rNTP) monomers are linked together to form a complementary RNA chain. Bases in the template DNA strand base-pair with complementary incoming rNTPs, which are then joined in a polymerization reaction catalyzed by RNA polymerase. The polymerization reaction involves a nucleophilic attack by the 3′ oxygen in the growing RNA chain on the α phosphate of the next nucleotide precursor to be added, which results in the formation of a phosphodiester bond and the release of pyrophosphate (PPi). As a consequence of this mechanism, RNA molecules are always synthesized in the 5′→3′ direction (Figure 1a).

Fig1. RNA is synthesized 5′→3′. (a) Polymerization of ribonucleotides by RNA polymerase during transcription. The ribonucleotide to be added at the 3′ end of a growing RNA strand is specified by base pairing between the next base in the template DNA strand and the complementary incoming ribonucleoside triphosphate (rNTP). A phosphodiester bond is formed when RNA polymerase catalyzes a reaction between the 3′ oxygen of the growing strand and the α phosphate of a correctly base-paired rNTP. RNA strands are always synthesized in the 5′→3′ direction and are opposite in polarity to their template DNA strands. (b) Conventions for describing RNA transcription. Top: The DNA nucleotide where RNA polymerase begins transcription is designated +1. The direction the polymerase travels on the DNA is “downstream,” and downstream bases are marked with positive numbers. The opposite direction is “upstream,” and upstream bases are marked with negative numbers. Some important gene features lie up stream of the transcription start site, including the promoter sequence that localizes RNA polymerase to the gene. Bottom: The DNA strand that is being transcribed is the template strand; its complement is the nontemplate strand. The RNA being synthesized is complementary to the template strand and is therefore identical with the nontemplate strand sequence, except with uracil in place of thymine.

The energetics of the polymerization reaction strongly favor the addition of ribonucleotides to the growing RNA chain because the high-energy bond between the α and β phosphates of rNTP monomers is replaced by the lower energy phosphodiester bond between nucleotides. The equilibrium for the reaction is driven further toward chain elongation by pyrophosphatase, an enzyme that catalyzes cleavage of the released PPi into two molecules of inorganic phosphate. Like the two strands in DNA, the template DNA strand and the growing RNA strand that is base-paired to it have opposite 5′→3′ directionality.

By convention, the site on the DNA template at which RNA polymerase begins transcription is numbered +1 (Figure 5-10b). Downstream denotes the direction in which a template DNA strand is transcribed; upstream denotes the opposite direction. Nucleotide positions in the DNA sequence downstream from a start site are indicated by a positive (+) sign; those upstream, by a negative (−) sign. Because RNA is synthesized 5′→3′, RNA polymerase moves down the template DNA strand in a 3′→5′ direction. The newly synthesized RNA is complementary to the template DNA strand; therefore, it is identical to the nontemplate DNA strand, with uracil in place of thymine.

Stages in Transcription To carry out transcription, RNA polymerase performs several distinct functions, as depicted in Figure 2. During transcription initiation, RNA polymerase, with the help of initiation factors (discussed later), recognizes and binds to a specific sequence of double stranded DNA called a promoter (step 1). After binding, RNA polymerase and the initiation factors separate the DNA strands to make the bases in the template strand available for base pairing with the bases of the rNTPs that it will polymerize (step 2 ). Approximately 12–14 base pairs of DNA around the transcription start site on the template strand are separated, which allows the template strand to enter the active site of the enzyme. The active site is where catalysis of phosphodiester bond formation between rNTPs that are complementary to the template strand takes place. The 12–14-base-pair region of melted DNA in the polymerase is known as the transcription bubble. Transcription initiation is considered complete when the first two ribonucleotides of an RNA chain are linked by a phosphodiester bond (step 3).

Fig2. Three stages in transcription. During initiation of transcription, RNA polymerase forms a transcription bubble and begins polymerization of ribonucleotides (rNTPs) at the start site, which is located within the promoter region. Once a DNA region has been transcribed, the separated strands reassociate into a double helix. The nascent RNA is displaced from its template strand except at its 3′ end. The 5′ end of the RNA strand exits the RNA polymerase through a channel in the enzyme. Termination occurs when the polymerase encounters a specific termination sequence (stop site). See the text for details. For simplicity, the diagram depicts transcription of four turns of the DNA helix encoding some 40 nucleotides of RNA. Most RNAs are considerably longer, requiring transcription of a longer region of DNA.

After several ribonucleotides have been polymerized, RNA polymerase dissociates from the promoter DNA and initiation factors (called σ-factors in bacteria, and general transcription factors in archaea and eukaryotes). During the strand elongation stage, RNA polymerase moves along the template DNA, opening the double-stranded DNA in front of its direction of movement and guiding the strands back together so that they reassociate at the upstream end of the transcription bubble (step 4). One ribonucleotide at a time is added by the polymerase to the 3′ end of the growing (nascent) RNA chain. During strand elongation, the enzyme maintains a melted region of approximately 14–20 base pairs in the transcription bubble. Approximately eight nucleotides at the 3′ end of the growing RNA strand remain base-paired to the template DNA strand in the transcription bubble. The elongation complex, comprising RNA polymerase, template DNA, and the nascent RNA strand, is extraordinarily stable. For example, RNA polymerase transcribes the longest known mammalian gene, containing about 2 million base pairs, without dissociating from the DNA template or re leasing the nascent RNA. RNA synthesis occurs at a rate of about 1000–2000 nucleotides per minute at 37 °C, so the elongation complex must remain intact for more than 24 hours to ensure continuous synthesis of pre-mRNA from this very long gene.

During transcription termination, the final stage in RNA synthesis, the completed RNA molecule is released from the RNA polymerase and the polymerase dissociates from the template DNA (step 5). Once it is released, an RNA polymerase is free to transcribe the same gene again or another gene.

Structure of RNA Polymerases The RNA polymerases of bacteria, archaea, and eukaryotic cells are fundamentally similar in structure and function. Bacterial RNA polymerases are composed of two related large subunits (β′ and β), two copies of a smaller subunit (α), and one copy of a fifth subunit (ω) that is not essential for transcription or cell viability, but that stabilizes the enzyme and assists in the assembly of its subunits. Archaeal and eukaryotic RNA polymerases have several additional small subunits associated with this core complex, which we describe in Chapter 9. Schematic diagrams of the transcription process generally show RNA polymerase bound to an unbent DNA molecule, as in Figure 2. However, x-ray crystallography and other studies of an elongating bacterial RNA polymerase indicate that the DNA bends at the transcription bubble (Figure 3).

Fig3. Bacterial RNA polymerase. This structure corresponds to the polymerase molecule in the elongation stage (step 4) of Figure 5-11. In this diagram, transcription is proceeding in the right ward direction. Arrows indicate where downstream DNA enters the polymerase and upstream DNA exits at an angle from the downstream DNA. The template strand is light violet, the nontemplate strand, dark violet; the nascent RNA, red. The RNA polymerase β′ subunit is gold; the β subunit, light yellow; and the α subunits visible from this angle, brown. Nucleotides complementary to the template DNA are added to the 3′ end of the nascent RNA strand on the right side of the transcription bubble. The newly synthesized nascent RNA exits the polymerase at the upstream side through a channel formed by the β subunit. The ω subunit is also visible from this angle. [Data courtesy of Seth Darst; see N. Korzheva et al., 2000, Science 289:619–625, and N. Opalka et al., 2003, Cell 114:335–345.]

Organization of Genes Differs in Prokaryotic and Eukaryotic DNA

 Having outlined the process of transcription, we now briefly consider the large-scale arrangement of information in DNA and how this arrangement dictates the requirements for RNA synthesis so that information transfer goes smoothly. In recent years, sequencing of entire genomes from multiple organisms has revealed not only large variations in the number of protein-coding genes, but also differences in their organization in bacteria and in eukaryotes.

The most common arrangement of protein-coding genes in bacteria has a powerful and appealing logic: genes encoding proteins that function together—for example, the enzymes required to synthesize the amino acid tryptophan— are most often found in a contiguous array in the DNA. Such an arrangement of genes in a functional group is called an operon because it operates as a unit from a single promoter. Transcription of an operon produces a continuous strand of mRNA that carries the message for a related series of proteins (Figure 4a). Each section of the mRNA represents the unit (or gene) that encodes one of the proteins in the series. This arrangement results in the coordinate expression of all the genes in the operon. Every time an RNA polymerase molecule initiates transcription at the promoter of the operon, all the genes of the operon are transcribed and translated. In prokaryotic DNA the genes are closely packed with very few noncoding gaps, and the DNA is transcribed directly into mRNA. Because DNA is not sequestered in a nucleus in prokaryotes, ribosomes have immediate access to the translation start sites in the mRNA as they emerge from the surface of the RNA polymerase. Consequently, translation of the mRNA begins even while the 3′ end of the mRNA is still being synthesized at the active site of the RNA polymerase.

Fig4. Gene organization in prokaryotes and in eukaryotes. (a) The tryptophan (trp) operon is a continuous segment of the E. coli chromosome containing five genes (blue) that encode the enzymes necessary for the stepwise synthesis of tryptophan. The entire operon is transcribed from one promoter into one long continuous trp mRNA (red). Translation of this mRNA begins at five different start sites, yielding five proteins (green). The order of the genes in the bacterial genome parallels the sequential function of the encoded proteins in the tryptophan synthesis pathway. (b) The five genes encoding the enzymes required for tryptophan synthesis in baker’s yeast (Saccharomyces cerevisiae) are carried on four different chromosomes. Each gene is transcribed from its own promoter to yield a primary transcript that is processed into a functional mRNA encoding a single protein. The lengths of the various chromosomes are given in kilobases (10³ bases).

This economical clustering of genes devoted to a single metabolic function is rarely found in eukaryotes, even simple ones such as yeasts, which can be metabolically similar to bacteria. Rather, eukaryotic genes encoding proteins that function together are most often physically separated in the DNA; indeed, such genes are usually located on different chromosomes. Each gene is transcribed from its own promoter, producing one mRNA, which is generally translated to yield a single polypeptide (Figure 4b).

Early research on the structure of eukaryotic genes in volved studies of viruses that infect animals. When re searchers analyzed the regions of a viral DNA molecule that encode viral mRNAs, they were surprised to observe that the sequence of a single viral mRNA was encoded in several regions of the viral DNA separated by DNA sequences that are not present in the mRNA. Later, the development of gene cloning and DNA sequencing allowed researchers to compare the genomic DNA sequences of multicellular organisms with the sequences of their mRNAs. This research revealed that most cellular mRNAs are also encoded in several separate regions of genomic DNA, called exons, separated by sequences of DNA called introns. Further studies showed that a gene is first transcribed into a long primary transcript that includes both exon sequences and the intron sequences that separate them. Subsequently, the introns are removed and the exons are spliced together. Although introns are common in multi cellular eukaryotes, they are extremely rare in bacteria and archaea and uncommon in many unicellular eukaryotes, such as baker’s yeast.

Eukaryotic Precursor mRNAs Are Processed to Form Functional mRNAs

In bacterial cells, which have no nuclei, translation of an mRNA into protein can begin at the 5′ end of the mRNA even while the 3′ end is still being synthesized by RNA polymerase. In other words, transcription and translation occur concurrently in bacteria. In eukaryotic cells, however, the site of RNA synthesis—the nucleus—is separated from the site of translation—the cytoplasm. Furthermore, the primary transcripts of protein-coding genes are precursor mRNAs (pre-mRNAs) that must undergo several modifications, collectively termed RNA processing, to yield a functional mRNA. This mRNA then must be exported to the cytoplasm before it can be translated into protein. Thus transcription and translation cannot occur concurrently in eukaryotic cells.

All eukaryotic pre-mRNAs are initially modified at the two ends, and these modifications are retained in mRNAs. As the 5′ end of a nascent RNA chain emerges from the surface of RNA polymerase, it is immediately acted on by several enzymes that together synthesize the 5′ cap, a 7-methylguanylate that is connected to the terminal nucleotide of the RNA by an unusual 5′,5′ triphosphate linkage (Figure 5). The cap protects an mRNA from enzymatic degradation and assists in its export to the cytoplasm. The cap is also bound by a protein factor required to begin translation in the cytoplasm.

Fig5. Structure of the 5′ methylated cap. The distinguishing chemical features of the 5′ methylated cap on eukaryotic mRNA are (1) the 5′→5′ linkage of 7-methylguanylate to the initial nucleotide of the mRNA molecule and (2) the methyl group on the 2′ hydroxyl of the ribose of the first nucleotide (base 1). Both of these features occur in all animal cells and in cells of higher plants; yeasts lack the methyl group on nucleotide 1. The ribose of the second nucleotide (base 2) is also methylated in vertebrates. See A. J. Shatkin, 1976, Cell 9:645.

Processing at the 3′ end of a pre-mRNA involves cleavage by an endonuclease to yield a free 3′-hydroxyl group, to which a string of adenylic acid residues is added one at a time by an enzyme called poly(A) polymerase. The resulting poly(A) tail contains 100–250 bases, being shorter in yeasts and invertebrates than in vertebrates. Poly(A) polymerase is part of a complex of proteins that can locate and cleave a transcript at a specific site and then add the correct number of A residues, in a process that does not require a template. As discussed further in Section 5.4 and in Chapter 10, the poly(A) tail has important functions both in translation of mRNA and in stabilizing pre mRNAs in the nucleus and fully processed mRNAs in the nucleus and cytoplasm.

Another step in the processing of many different eukaryotic mRNA molecules is RNA splicing: the internal cleavage of a transcript to excise the introns and stitch together the coding exons. Figure 6 summarizes the basic steps in eukaryotic mRNA processing using the β-globin gene as an example.

Fig6. Overview of RNA processing. RNA processing produces functional mRNA in eukaryotes. The β-globin gene contains three protein-coding exons (constituting the coding region) and two intervening noncoding introns. The introns interrupt the protein- coding sequence between the codons for amino acids 31 and 32 and 105 and 106. Transcription of eukaryotic protein-coding genes starts before the sequence that encodes the first amino acid and extends beyond the sequence that encodes the last amino acid, resulting in noncoding regions at the ends of the primary transcript. These untranslated regions (UTRs) are retained during processing. The 5′ cap (m7Gppp) is added during formation of the primary RNA transcript, which extends beyond the poly(A) site. After cleavage at the poly(A) site and addition of multiple A residues to the 3′ end, splicing removes the introns and joins the exons. The small numbers refer to positions in the 147–amino acid sequence of β-globin.

The functional eukaryotic mRNAs produced by RNA processing retain noncoding regions, referred to as untranslated regions (UTRs), at each end. In mammalian mRNAs, the 5′ UTR may be a hundred or more nucleotides long, and the 3′ UTR may be several kilobases in length. Bacterial mRNAs also usually have 5′ and 3′ UTRs, but these regions are much shorter than those in eukaryotic mRNAs, generally containing fewer than 10 nucleotides. As discussed in Chapter 10, the 5′ UTR and 3′ UTR sequences participate in regulation of mRNA translation and stability, and 3′ UTRs also function in the localization of many mRNAs to specific regions of the cytoplasm.

Alternative RNA Splicing Increases the Number of Proteins Expressed from a Single Eukaryotic Gene

In contrast to bacterial and archaeal genes, the vast majority of genes in multicellular eukaryotes contain multiple introns. As noted in Chapter 3, many proteins from higher eukaryotes have a multidomain tertiary structure. Individual repeated protein domains are often encoded by one exon or by a small number of exons that are repeated in genomic DNA and encode identical or nearly identical amino acid sequences. Such repeated exons are thought to have evolved from multiple duplications of a length of DNA lying between two sites in introns on either side of an exon, resulting in insertion of a string of repeated exons separated by introns. The presence of multiple introns in many eukaryotic genes permits expression of multiple, related proteins from a single gene by means of alternative splicing. In higher eukaryotes, alternative splicing is an important mechanism for production of different forms of a protein, called isoforms, by different types of cells.

Fibronectin, a multidomain protein found in mammals, provides a good example of alternative splicing (Figure 7). Fibronectin is a long, adhesive protein secreted into the extracellular space that can bind other proteins together. What and where it binds depends on which domains are spliced together. The fibronectin gene contains numerous exons, grouped into several regions corresponding to specific domains of the protein. Fibroblasts produce fibronectin mRNAs that contain exons EIIIA and EIIIB; these exons encode a protein domain that binds tightly to proteins in the fibroblast plasma membrane. Consequently, this fibronectin isoform adheres fibroblasts to the extracellular matrix. Alternative splicing of the fibronectin primary transcript in hepatocytes, the major type of cell in the liver, yields mRNAs that lack the EIIIA and EIIIB exons. As a result, the fibronectin secreted by hepatocytes into the blood does not adhere tightly to fibroblasts or to most other cell types, which al lows it to circulate. During formation of blood clots, however, other fibrin-binding domains of hepatocyte fibronectin bind to fibrin, one of the principal constituents of blood clots. Yet another domain of the bound fibronectin then interacts with integrins on the membranes of passing platelets, thereby expanding the clot by addition of platelets.

Fig7. Alternative splicing. The ∼75-kb fibronectin gene (top) contains multiple exons; splicing of the fibronectin transcript varies by cell type. The EIIIB and EIIIA exons (green) encode binding domains for specific proteins on the surface of fibroblasts. The fibronectin mRNA produced in fibroblasts includes the EIIIA and EIIIB exons, whereas these exons are spliced out of fibronectin mRNA in hepatocytes. In this diagram, introns (black lines in the top diagram of the fibronectin gene) are not drawn to scale; most of them are much longer than any of the exons.

More than 20 different isoforms of fibronectin have been identified, each encoded by a different, alternatively spliced mRNA composed of a unique combination of fibronectin gene exons. Sequencing of large numbers of mRNAs isolated from various tissues and comparison of their sequences with genomic DNA has revealed that nearly 90 percent of all human genes are expressed as alternatively spliced mRNAs. Clearly alternative RNA splicing greatly expands the number of proteins encoded by the genomes of higher, multicellular organisms.

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مواضيع ذات صلة

Nonstandard Base Pairing Often Occurs Between Codons and Anticodons

The Folded Structure of tRNA Promotes Its Decoding Functions

Messenger RNA Carries Information from DNA in a Three-Letter Genetic Code

DNA Can Undergo Reversible Strand Separation

Native DNA Is a Double Helix of Complementary Antiparallel Strands

A Nucleic Acid Strand Is a Linear Polymer with End-to-End Directionality

Abnormalities of Chromosome Structure

Impact of Genomic Variation

Variation in individual Genomes

Variation in gene Expression and its relevance to medicine

Allelic Imbalance in gene expression

Gene expression as the integration of Genomic and Epigenomic signals