The process of RNA synthesis in bacteria—depicted in Figure 1—is cyclical and involves multiple steps. First RNA polymerase holoenzyme (Eσ) must locate and then specifically bind a promoter (P; see Figure 1). Once the promoter is located, the Eσ–promoter DNA complex undergoes a temperature-dependent conformational change and unwinds, or melts the DNA in and around the TSS (at +1). This complex is termed the preinitiation complex (PIC). Unwinding allows the active site of the Eσ to access the template strand, which of course dictates the sequence of ribonucleotides to be polymerized into RNA. The first DNA template-directed nucleotide (typically, though not always a purine) then associates with the nucleotide-binding site of the enzyme, and in the presence of the next appropriate nucleotide bound to the polymerase, RNAP catalyzes the formation of the first phosphodiester bond, and the nascent chain is now attached to the polymerization site on the β subunit of RNAP. This reaction is termed initiation. The nascent dinucleotide retains the 5′-triphosphate of the initiating nucleotide (in Figure 1 this happens to be ATP).

Fig1. 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.

RNA polymerase continues to incorporate nucleotides +3 to ~+10, at which point the polymerase undergoes another conformational change and moves away from the promoter; this reaction is termed promoter clearance. On many genes the σ–factor dissociates from the ββ′α2 assembly at this point. The elongation phase then commences, and here the nascent RNA molecule grows 5′–>3′ as consecutive NTP incorporation steps continue cyclically, antiparallel to the template. The enzyme polymerizes the ribonucleotides in the specific sequence dictated by the template strand and interpreted by Watson-Crick base-pairing rules. Pyrophosphate (PPi ) is released following each cycle of polymerization. As for DNA synthesis, the liberated PPi is rapidly degraded to two molecules of inorganic phosphate (Pi ) by ubiquitous pyro phosphatases, thereby providing irreversibility on the overall synthetic reaction. The decision to stay at the promoter in a poised or stalled state, or transition to elongation can be an important regulatory step in both prokaryotic and eukaryotic mRNA gene transcription.

As the elongation complex containing RNA polymerase progresses along the DNA molecule, DNA unwinding must occur in order to provide access for the appropriate base pairing to the nucleotides of the coding strand. The extent of this transcription bubble (ie, DNA unwinding) is constant throughout transcription and has been estimated to be about 20 bp per polymerase molecule (see Figures 2; 1). Thus, the size of the unwound DNA region is dictated by the polymerase and is independent of the DNA sequence in the com plex. RNA polymerase has an intrinsic “unwindase” activity that opens the DNA helix (see PIC formation earlier). The fact that the DNA double helix must unwind, and the strands separate at least transiently for transcription implies some temporary disruption of the nucleosome structure of eukaryotic cells. Topoisomerase both precedes and follows the progressing RNA polymerase to prevent the formation of superhelical tension that would serve to increase the energy required to unwind the template DNA ahead of RNAP.

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.

Termination of the synthesis of RNA in bacteria is signaled by sequences in the template DNA (see Figure 1; T) and sequences within the transcript. On many genes RNAP alone efficiently terminates transcription. However, on a sub set of genes a termination protein termed rho (ρ) factor is required to mediate transcription termination. After termination both free core RNAP (E) and product RNA dissociate from the DNA template. The resulting free core RNAP (E) is able to associate with σ-factor to reform Eσ, and re-enter the transcription cycle. In eukaryotic cells, termination is less well understood; however, the proteins catalyzing RNA processing, termination, and polyadenylation all appear to load onto RNA polymerase II soon after initiation. In both prokaryotes and eukaryotes, multiple RNA polymerase molecules typically transcribe the same template strand of a gene simultaneously, but the process is phased and spaced in such a way that at any one moment each is transcribing a different portion of the DNA sequence (see Figures 3 and 4).

Fig3. 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.

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.

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مواضيع ذات صلة

Eukaryotic Promoters Are More Complex

Bacterial Promoters Are Relatively Simple

RNA is Synthesized From A DNA Template by RNA Polymerases

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