The processes of DNA and RNA synthesis are similar in that they involve (1) the general steps of initiation, elongation, and termination with 5′–3′ polarity; (2) large, multicomponent initiation and polymerization complexes; and (3) adherence to Watson-Crick base-pairing rules. However, DNA and RNA synthesis do differ in several important ways, including the following: (1) ribonucleotides are used in RNA synthesis rather than deoxyribonucleotides; (2) in RNA U replaces T as the complementary base for A; (3) a primer is not involved in RNA synthesis because RNA polymerases have the intrinsic ability to initiate synthesis de novo; (4) in a given cell only portions of the genome are vigorously transcribed or copied into RNA, at any given time/condition, whereas the entire genome must be copied, once and only once during DNA replication; and (5) there is no highly active, efficient proofreading function during transcription.

The process of synthesizing RNA from a DNA template has been characterized best in prokaryotes. Although in mammalian cells, aspects of the regulation of RNA synthesis and the processing of the RNA transcripts are different from those in prokaryotes, the process of RNA synthesis per se is quite similar in these two classes of organisms. Indeed, there is significant amino acid sequence conservation between prokaryotic and eukaryotic DNA-dependent RNA polymerases. Consequently, the description of RNA synthesis in prokaryotes, where it is best understood, is applicable to eukaryotes even though the enzymes involved and the regulatory signals, though related, are different.

The Template Strand of DNA Is Transcribed, Or Copied Into RNA

The sequence of ribonucleotides in an RNA molecule is complementary to the sequence of deoxyribonucleotides in one strand of the double-stranded DNA molecule. The strand that is transcribed or copied into an RNA molecule is referred to as the template strand of the DNA. The other DNA strand, the non-template strand, is frequently referred to as the coding strand of that gene. It is called this because, with the exception of T for U changes, it corresponds exactly to the sequence of the mRNA primary transcript, which encodes the (protein) product of the gene. In the case of a double stranded DNA molecule containing many genes, the template strand for each gene will not necessarily be the same strand of the DNA double helix (Figure 1). Thus, a given strand of a double-stranded DNA molecule will serve as the template strand for some genes and the coding strand of other genes. Note that the nucleotide sequence of an RNA transcript will be the same (except for U replacing T) as that of the coding strand. The information in the template strand is read out in the 3′–5′ direction. Though not shown in Figure 1, there are instances of genes embedded within other genes.

Fig1. Genes can be transcribed from both strands of DNA. The arrowheads indicate the direction of transcription (polarity). Note that the template strand is always read in the 3′–5′ direction. The opposite strand is called the coding strand because it is identical (except for T for U changes) to the mRNA transcript (the primary transcript in eukaryotic cells) that encodes the protein product of the gene.

DNA-Dependent RNA Polymerase Binds to a Distinct Site, the Promoter, & Initiates Transcription

 DNA-dependent RNA polymerase (RNAP) is the enzyme responsible for the polymerization of ribonucleotides into a sequence complementary to the template strand of the gene (Figures 2 and 3). A key aspect of the process of RNA synthesis is how RNAP identifies and binds template DNA at the correct genomic location. The enzyme attaches at a specific site—the promoter—on the DNA template. This is followed by unwinding of the DNA duplex at, and immediately downstream of the promoter. Once RNAP is specifically bound and has unwound the DNA template at the promoter, initiation of RNA synthesis commences at the transcription start site (TSS) with the formation of an initial dinucleotide, the sequence of which is dictated by the DNA sequence of the template strand. The process of phosphodiester bond formation continues until a termination sequence is reached (see Figure 3). A transcription unit is defined as that region of DNA that includes the signals for transcription initiation, elongation, and termination. The RNA product, which is synthesized in the 5′–3′ direction, is the primary transcript. Transcription frequency varies from gene to gene but can be quite high. An electron micrograph of transcription in action is presented in Figure 4. In prokaryotes, a transcription unit can represent the product of several contiguous genes, while mammalian cell transcription units usually contain but a single gene. The 5′ termini of the primary RNA transcript and the mature cytoplasmic RNA are identical in bacteria. Thus, the bacterial TSS corresponds to the 5′ nucleotide of the mRNA. This is designated position +1, as is the corresponding nucleotide in the DNA. The numbering system of the transcription unit increases positively as the sequence proceeds downstream from the start site. This convention makes it easy to locate particular regions, or functional elements of transcripts (and their encoded proteins if protein coding), such as intron and exon boundaries; translation start/stop signals etc. The nucleotide in the promoter adjacent to the transcription initiation site in the upstream direction is designated −1, and these negative numbers increase as the sequence proceeds upstream, away from the TSS. This +/− numbering system provides a conventional way of defining the location of regulatory elements in a gene (Figure 5).

Fig2. RNA polymerase catalyzes the polymerization of ribonucleotides into an RNA sequence that is complementary to the template strand of the gene. The RNA transcript has the same polarity (5′–3′) as the coding strand but contains U rather than T. Bacterial RNAP consists of a core complex of two β subunits (β and β′) and two α subunits. The holoenzyme form of RNA polymerase contains the σ subunit bound to the α2 ββ′ core assembly. The ω subunit is not shown. The transcription “bubble” is an approximately 20-bp area of melted DNA, and the entire complex covers 30 to 75 bp of DNA depending on the conformation of RNAP.

Fig3. The transcription cycle. The transcription cycle can be described in six steps: (1) Template binding and closed RNA polymerase-promoter complex formation: RNAP binds to DNA and then locates a promoter (P) DNA sequence element. (2) Open promoter complex formation: Once bound to the promoter, RNAP melts the two DNA strands to form an open promoter complex; this complex is also referred to as the preinitiation complex or PIC. Strand separation allows the polymerase to access the coding information in the template strand of DNA. (3) Chain initiation: Using the coding information of the template, RNAP catalyzes the coupling of the first base (often a purine) to the second, template-directed ribonucleoside triphosphate to form a dinucleotide (in this example forming the dinucleotide 5′ pppApNOH 3′). (4) Promoter clearance: After RNA chain length reaches ~10 to 20 nt, the polymerase undergoes a conformational change and then is able to move away from the promoter, transcribing down the transcription unit. On many genes σ-factor is released from RNAP at this phase of the transcription cycle. (5) Chain elongation: Successive residues are added to the 3′-OH terminus of the nascent RNA molecule until a transcription termination DNA sequence element (T) is encountered by RNAP. (6) Chain termination and RNAP release: Upon encountering the transcription termination site, RNAP undergoes an additional conformational change that leads to release of the completed RNA chain, the DNA template, and RNAP. Following reformation of holoenzyme (Eσ), RNAP can rebind to DNA beginning the promoter search process and the cycle is repeated. Note that all of the steps in the transcription cycle are facilitated by additional proteins, and indeed are often subjected to regulation by positive and/or negative-acting factors.

Fig4. Schematic representation of an electron photomicrograph of tandem arrays of amphibian rRNA-encoding genes in the process of being transcribed. The magnification is about 6000×. Note that the length of the transcripts increases as the RNA polymerase molecules progress along the individual rRNA genes from transcription start sites (filled circles) to transcription termination sites (open circles). RNA polymerase I (not visualized here) is at the base of the nascent rRNA transcripts. Thus, the proximal end of the transcribed gene has short transcripts attached to it, while much longer transcripts are attached to the distal end of the gene. The arrows indicate the direction (5′→3′) of transcription.

Fig5. Prokaryotic promoters share two regions of highly conserved nucleotide sequence. These regions are located 35- and 10-bp upstream of the TSS, which is indicated as +1. By convention, all nucleotides upstream of the transcription initiation site (at +1) are numbered in a negative sense and are referred to as 5′-flanking sequences, while sequences downstream of the +1 TSS are numbered in a positive sense. Also, by convention, the promoter DNA regulatory sequence elements such as the −35 and the −10 TATAAT elements are described in the 5′→3′ direction and as being on the coding strand. These elements function only in double-stranded DNA. Other transcriptional regulatory elements, however, can often act in a direction independent fashion, and such cis-elements are drawn accordingly in any schematic. Note that the transcript produced from this transcription unit has the same polarity or “sense” (ie, 5′→3′ orientation) as the coding strand. Termination determining cis-elements reside at the end of the transcription unit. By convention, the sequences downstream of the site at which transcription termination occurs are termed 3′-flanking sequences.

The primary transcripts generated by RNA polymerase II— one of the three distinct nuclear DNA-dependent RNA polymerases in eukaryotes—are promptly modified by the addition of 7-methylguanosine triphosphate caps, which persist and eventually appear on the 5′ end of mature cytoplasmic mRNA. These caps are necessary for the subsequent processing of the primary transcript to mRNA, for the translation of the mRNA, and for protection of the mRNA against nucleolytic attack by 5′-exonucleases.

Bacterial DNA-Dependent RNA Polymerase Is a Multisubunit Enzyme

 The basic DNA-dependent RNA polymerase of the bacterium Escherichia coli exists as an approximately 400-kDa core complex consisting of two identical α subunits, two large β and β′ subunits, and an ω subunit. The β subunit binds Mg2+ions and composes the catalytic subunit (see Figure 2).

The core RNA polymerase, ββ′α2 ω, often termed E, associates with a specific protein factor (the sigma [σ] factor) to form holoenzyme, ββ′α2 σω, or Eσ. The genes encoding all these proteins are essential for viability with an exception of ω-encoding gene. The σ subunit enables the core enzyme to recognize and bind the promoter region (see Figure 5) to form the preinitiation complex (PIC). There are multiple, distinct σ-factor encoding genes in all bacterial species. Sigma factors have a dual role in the process of promoter recognition: σ association with core RNA polymerase decreases its affinity for nonpromoter DNA, while simultaneously increasing holoenzyme affinity for promoter DNA. Within the bacterial cell these multiple σ-factors compete for interaction with limiting core RNA polymerase (ie, E). Each of these unique σ-factors act as a regulatory protein that modifies the promoter recognition specificity of the resulting unique RNA polymerase holoenzyme (ie, Eσ1 , Eσ2 ,…). The appearance of different σ-factors and their association with core RNA polymerase to form novel holoenzyme forms Eσ1 , Eσ2 ,…, can be correlated temporally with various programs of gene expression in prokaryotic systems such as sporulation, growth in various poor nutrient sources, and the response to heat shock.

Mammalian Cells Possess Three Distinct Nuclear DNA-Dependent RNA Polymerases & a Single DNA-Dependent Mitochondrial RNA Polymerase

 Some of the distinguishing properties of mammalian nuclear RNA polymerases are described in Table 1. Each of these DNA-dependent RNA polymerases is responsible for transcription of different sets of genes. The sizes of the RNA polymerases range from MW 500 kDa to 600 kDa, and the enzymes exhibit more complex subunit profiles than prokaryotic RNA polymerases. They all have two large subunits, which remarkably bear strong sequence and structural similarities to prokaryotic β and β′ subunits, and a number of smaller subunits—as many as 14 in the case of RNA pol III. The functions of each of the subunits are not yet fully understood. A pep tide toxin from the mushroom Amanita phalloides, α-amanitin, is a specific differential inhibitor of the eukaryotic nuclear DNA-dependent RNA polymerases and as such has proved to be a powerful research tool (Table 1). α-Amanitin blocks the translocation of RNA polymerase during phosphodiester bond formation. Mitochondria contain a dedicated DNA dependent RNA polymerase (mtRNAP) that is encoded by a nuclear gene. This mtRNAP, in concert with two accessory initiation and one termination factor, is responsible for all mtDNA gene transcription.

Table1. Nomenclature & Properties of Mammalian Nuclear DNA-Dependent RNA Polymerases

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مواضيع ذات صلة

DNA & Chromosome Integrity Is Monitored Throughout the Cell Cycle

All Organisms Contain Elaborate Evolutionarily Conserved Mechanisms to Repair Damaged DNA

Micro-RNA and Long Noncoding RNA

Histone Organization

Noncoding DNA

DNA Synthesis Occurs During the S Phase of the Cell Cycle

Formation of Replication Bubbles

Initiation & Elongation of DNA Synthesis

The Exact Function of Much of the Mammalian Genome is Not Well Understood

DNA is organized into Chromosomes

Some Regions of Chromatin are “Active” & Others are “Inactive”

The Nucleosome Contains Histone & DNA