As well as having global roles in storing and transmitting genetic information and sup porting chromosome function, DNA can have cell-type-specific functions because it contains sequences that can be used to make RNA and polypeptides in ways that differ from one cell type to another. Genes are discrete DNA segments that are spaced at irregular intervals along a DNA strand and serve as templates for making complementary RNA sequences (transcription). The initial primary RNA transcript must then undergo a series of maturation steps that ultimately result in a mature, functional noncoding RNA or a messenger RNA that will in turn serve as a template to make a polypeptide. Some of the gene products are needed by essentially all cells for a variety of vital cell processes (DNA replication, protein synthesis, and so on). But other RNA and protein products are made in some cell types but not others and may even be specific for individual cells in some exceptional cases (for example, individual B and T lymphocytes can make cell specific immunoglobulins and T-cell receptors, respectively).
The DNA compositions of the different cell types in a multicellular organism are essentially identical. The variation between cells happens because of differences in gene expression, primarily at the level of transcription: different genes are transcribed in different cells according to the needs of the cells. Some genes, known as housekeeping genes, need to be expressed in essentially all cells, but other genes show tissue- specific gene expression or they may be expressed at specific times (for example, at specific stages of development or of the cell cycle).
The basic process of transcription
We consider the details of transcription more fully in Chapter 10 when we consider how gene expression is regulated. Here, we are concerned with the most basic features of transcription. At its most fundamental level, transcription means that RNA is synthesized using DNA-directed RNA polymerases. That is, DNA strands serve as templates for RNA synthesis. In eukaryotic cells, the bulk of cellular RNA is synthesized in the nucleus by transcribing nuclear genes, but a small amount is synthesized in mitochondria and the chloroplasts of plant cells by transcribing the DNA found in these organelles.
During transcription the DNA helix needs to be unwound locally to allow the RNA polymerase to gain access to a separated DNA strand from which it will make a complementary RNA sequence. The RNA transcript has a complementary sequence to that of the template strand of the DNA, and has the same 5′ → 3′ direction and base sequence (except that U replaces T) as the other, nontemplate DNA strand. The nontemplate strand is often called the sense strand, and the template strand is often called the antisense strand (Figure 1).
Fig1. Transcribed RNA is complementary in sequence to one strand of DNA. During transcription, the DNA double helix is locally unwound as the RNA polymerase advances. One DNA strand, the template strand, is used by the polymerase as a template to synthesize a complementary RNA strand, and the RNA is synthesized from ribonucleoside triphosphate precursors (rNTPs). The polymerase cleaves rNTPs to give ribonucleoside monophosphates (rNMPs) that are inserted one nucleotide at a time by joining to the 3′ hydroxyl group of the previous nucleotide according to base-pairing rules. (However, the primary RNA transcript will have a triphosphate [PPP] group at its 5′ end that has not been cleaved.) The base sequence of the RNA transcript will be complementary in sequence to the template strand and will therefore be identical to the sense strand of DNA (except that U replaces T). During transcription, a very short region of DNA RNA double helix is formed transiently. As the polymerase advances, the area with the DNA–RNA helix advances behind it, with the DNA double helix re-forming behind that, displacing the RNA.
RNA polymerases synthesize RNA from four nucleotide precursors: ATP, CTP, GTP, and UTP. Elongation involves the addition of the appropriate ribonucleotide monophosphate residue (AMP, CMP, GMP, or UMP) to the free 3′ hydroxyl group at the 3′ end of the growing RNA strand. These nucleotides are derived by splitting a pyrophosphate residue (PPi) from their appropriate ribonucleoside triphosphate (rNTP) precursors. Note that the initiator nucleotide at the extreme 5′ end of a primary transcript retains its 5′ tri phosphate group.
In documenting gene sequences, it is customary to show only the DNA sequence of the sense strand. The orientation of sequences relative to a gene normally refers to the sense strand. For example, the 5′ end of a gene refers to sequences at the 5′ end of the sense strand, and upstream or downstream sequences flank the gene at its 5′ or 3′ ends, respectively, with reference to the sense strand.
For transcription to proceed efficiently, various proteins (transcription factors) must bind to particular DNA sequence elements (collectively called a promoter) that are often located close to and upstream of a gene. The bound transcription factors serve to position and guide the RNA polymerase. Additional DNA sequence elements and transcription factors are also important.
RNA polymerase classes in eukaryotic cells
There are four classes of DNA-dependent RNA polymerase in eukaryotic cells. As described below, three of them are large multisubunit enzymes that are used in transcribing nuclear genes. A distantly related, single-subunit RNA polymerase is devoted to transcribing mitochondrial DNA. Note that the mitochondrial RNA polymerase is, however, encoded by a nuclear gene: it makes an mRNA that is translated on cytoplasmic ribosomes and then imported into mitochondria.
Unlike DNA polymerases, RNA polymerases do not need an oligonucleotide primer to initiate the RNA synthesis, but the RNA polymerases cannot initiate transcription by themselves. Instead, protein regulators known as transcription factors must activate the process by binding to certain regulatory DNA sequence elements within the gene or its vicinity. A crucial regulatory element is the promoter, a collection of closely spaced, short DNA sequence elements in the immediate vicinity of a gene. Promoters are recognized and bound by transcription factors that then guide and activate the polymerase. Transcription factors are said to be trans-acting, because they are produced by remote genes and need to migrate to their sites of action. In contrast, promoter sequences are cis-acting, because they are located on the same DNA molecule as the genes they regulate.
RNA polymerase II and transcription of protein-coding genes in the nucleus
As an illustration of how the nuclear RNA polymerases work in cells, we take the important example of RNA polymerase II, which is responsible for transcribing all the protein coding genes in the nucleus plus many important genes encoding different noncoding RNAs (Table 1). It relies on both general transcription factors (operating in all cells and important for expressing genes in diverse types of cell) plus tissue-specific and sometimes even cell-specific factors (to permit some genes to be expressed in only certain types of cell).
Table1. THE FOUR CLASSES OF EUKARYOTIC RNA POLYMERASE
For a gene to be transcribed by RNA polymerase II, the DNA at the transcription initiation site must first be bound by general transcription factors, to form a pre- initiation complex. The complex that is required to initiate transcription by an RNA polymerase is known as the basal transcription apparatus and consists of the polymerase plus all of its associated general transcription factors. (Note that although there are fixed transcription initiation sites, termination of RNA polymerase II transcripts is not regulated at the DNA level, but instead depends on RNA processing).
General transcription factors required by RNA polymerase II include TFIIA (transcription factor for RNA polymerase II, complex A), TFIIB, TFIID, TFIIE, TFIIF, and TFIIH. Of these, TFIID and TFIIE are known to bind specific core sequence elements within the promoter, such as the elements with the consensus sequences given in Figure 2. Note that these transcription factors may themselves comprise a number of components. For example, TFIID consists of the TATA box-binding protein (TBP) plus various TBP-associated factors (TAF proteins) that regulate how TBP binds to the TATA box.
Fig2. Consensus sequences for some core promoter elements often found in genes transcribed by RNA polymerase II. The TATA box is bound by the TATA-binding protein (TBP) subunit of transcription factor TFIID. The initiator (Inr) element defines the transcription start site (the A highlighted in red) when located 25–30 base pairs (bp) from a TATA box. The downstream core promoter element (DPE) is only functional when placed precisely at +28 to +32 bp relative to the highlighted A of an Inr element. Both Inr and DPE are bound by TFIID. Transcription factor TFIIB binds to BRE (TFIIB recognition element) and accurately positions RNA polymerase at the transcription start site. However, none of these elements is either necessary or sufficient for promoter activity, and many active RNA polymerase II promoters lack all of them. N represents any nucleotide. (Modified with permission from Smale ST & Kadonaga JT [2003] Annu Rev Biochem 72:449–479; PMID 12651739. © 2003 by Annual Reviews, http://www. annualreviews.org.)
TFIIF regulates the interaction between RNA polymerase II and TBP and helps attract TFIIE so that TFIIH can be recruited. The latter performs key tasks. Notably, it unwinds the DNA at the transcription start point, and it activates RNA polymerase II, releasing it from the promoter.
In addition to the general transcription factors required by RNA polymerase II, specific recognition elements are recognized by tissue-restricted transcription factors. For example, an enhancer is a cluster of cis-acting short sequence elements that can enhance the transcriptional activity of a small subset of genes. Unlike a promoter, which has a relatively constant position with regard to the transcriptional initiation site, enhancers are located at variable (often considerable) distances from their transcriptional start sites. Furthermore, their function is independent of their orientation. Enhancers do, however, also bind gene regulatory proteins. The DNA between the promoter and enhancer sites loops out, which brings the two different DNA sequences together and allows the proteins bound to the enhancer to interact with the transcription factors bound to the promoter, or with the RNA polymerase.
A silencer has similar properties to an enhancer but it inhibits, rather than stimulates, the transcriptional activity of a specific gene.
Different sets of RNA genes are transcribed by the three eukaryotic RNA polymerases
The protein-coding genes in nuclear DNA are always transcribed by RNA polymerase II. However, nuclear RNA genes (genes that make functional noncoding RNA) may be transcribed by RNA polymerases I, II, or III, depending on the type of RNA (Table 1). RNA polymerase I is unusual because it is dedicated to transcribing RNA from a single transcription unit, generating a large transcript that is then processed to yield three types of ribosomal RNA.
RNA polymerase II synthesizes various types of small noncoding RNA in addition to mRNA. They include many types of small nuclear RNA (snRNA) and small nucleolar RNA (snoRNA) that are involved in different RNA processing events. In addition, it synthesizes many microRNAs (miRNAs) and long noncoding RNAs (lncRNAs) that can show tissue-specific expression and typically regulate expression of distinctive sets of target genes.
RNA polymerase III transcribes a variety of small noncoding RNAs that are typically expressed in almost all cells (Table1). In many cases, the genes are known to have internal promoters that lie downstream of the transcriptional start site (Figure3). Internal promoters are possible because the job of a promoter is simply to attract transcription factors that will guide the RNA polymerase to the correct transcriptional start site. By the time the polymerase is in place and ready to initiate transcription, any transcription factors previously bound to downstream promoter elements will be removed from the template strand (see Figure3).
Fig3. Internal promoter elements in many genes transcribed by RNA polymerase III. (A) tRNA genes have an internal promoter consisting of an A box (located within the D arm of the tRNA) and a B box that is usually found in the TψC arm of tRNA. The promoter of the Xenopus 5S rRNA gene has three components: an A box (+50 to +60), an intermediate element (IE; +67 to +72), and the C box (+80 to +90). Arrows mark the +1 position. (B) Transcription factor binding to allow expression of a tRNA gene. TFIIIC binds to the A and B boxes of the internal promoter of a tRNA gene then guides the binding of another transcription factor, TFIIIB, to a position upstream of the transcriptional start site. TFIIIB guides RNA polymerase III (Pol III) to bind to the transcriptional start site. Thereafter, TFIIIC is no longer required; any bound TFIIIC will subsequently be removed from the internal promoter to allow unhindered transcription.
