Snyder and Champness Molecular Genetics of Bacteria. Tina M. Henkin

Figure 2.7 Transcription begins at a promoter and ends at a transcription terminator. (A) The RNA polymerase core must bind σ factor to recognize a promoter. (B) Transcription begins when the strands of DNA are opened at the promoter, and the first ribonucleoside triphosphate (rNTP), usually ATP or GTP, enters the active site opposite the +1 nucleotide in the template strand. (C) As RNA polymerase moves along the DNA, polymerizing ribonucleotides into RNA (green), it forms a transcription bubble containing an RNA-DNA double-stranded hybrid, which helps to hold the RNA polymerase on the DNA. The sigma factor is released after RNA polymerase leaves the promoter, and transcription by the RNA polymerase core enzyme continues. (D) The RNA polymerase stops transcription, comes off the DNA, and releases the newly synthesized RNA at a transcription terminator.

      ISOMERIZATION

      When RNA polymerase holoenzyme first binds to the promoter, the DNA is double stranded. The resulting complex is called the RP_c, because the DNA strands are still “closed.” In the next step, the β′ pincer of the crab claw closes around the DNA to form the active-site channel around the template strand of the DNA. This allows the σ₂ region to separate the strands of DNA at the –10 region and bind to the nontemplate strand in a process called isomerization (Figures 2.8 and 2.9). Recall that AT base pairs are less stable than GC pairs, so the AT-rich –10 sequence is relatively easy to melt. The complex is now called the open complex (RP_o), since the strands of DNA at the –10 region of the promoter are “open.” The +1 nucleotide of the template strand is held in the active-site channel, where the polymerization reaction is about to occur.

      INITIATION

In the initiation process, a single nucleoside triphosphate (usually an ATP or guanosine triphosphate [GTP]) enters through the secondary channel and pairs with nucleotide + 1 (usually a T or C, respectively) in the template strand in the active site of the enzyme. Then, a second nucleoside triphosphate enters, and if that nucleoside triphosphate can base pair with the +2 nucleotide of the template strand, a phosphodiester bond forms between its α phosphate and the 3′ hydroxyl of the first nucleotide, releasing two phosphates in the form of pyrophosphate (Figure 2.5A). This is called the initiation complex or initial transcription complex and is the step at which the antibiotic rifampin can block transcription (see Box 2.1). Rifampin binds to RNA polymerase in the β-subunit face of the active-site channel in such a way that the growing RNA encounters it when it reaches a length of only 2 or 3 nucleotides, preventing further growth of the RNA chain and freezing the RNA polymerase on the promoter. This explains why rifampin blocks only the initiation of transcription.

Even in the absence of the antibiotic rifampin, the RNA polymerase is not yet free to continue transcription. When the RNA chain grows to a length of about 10 nucleotides, it encounters the σ_3.2 loop, which blocks the site in RNA polymerase through which the newly synthesized RNA will emerge, a region called the exit channel (Figure 2.11). This causes transcription to stop, often releasing a short transcript about 10 nucleotides in length. This process is called abortive initiation and occurs to various degrees on many promoters for reasons that are not well understood. Eventually, a growing (or nascent) transcript pushes the σ_3.2 loop aside and enters the exit channel, usually causing the σ factor to be released from the core RNA polymerase. At this point, designated promoter escape, RNA polymerase has left the promoter site and has entered the elongation phase, during which the transcription bubble is enlarged to 17 bp, the complex is stabilized, and synthesis of RNA proceeds efficiently as the enzyme moves along the DNA template.

Figure 2.8 Overview of transcription. (A) The transcription cycle. Each step is discussed separately in the text. (B) Summary of the major steps in transcription initiation. R, RNA polymerase; P, promoter DNA; AP, abortive products; dsDNA, double-stranded DNA. Modified from Geszvain K, Landick R, in Higgins NP (ed), The Bacterial Chromosome (ASM Press, Washington, DC, 2005).

Figure 2.9 Transcription initiation. (A) Binding of σ to RNA polymerase core. (B) RNA polymerase holoenzyme binds to promoter DNA. (C) The initial RNA polymerase-promoter complex contains fully double-stranded DNA and is called the closed complex (RP_C). (D) The RNA polymerase-promoter complex isomerizes to form the open complex (RP_O).

      ELONGATION

Figure 2.12 shows the transcription elongation complex (TEC) in the process of elongating the RNA transcript. Most of the features are mentioned above, including the approximately 17-bp transcription bubble where the two strands of DNA are separated, the approximately 8- to 9-bp RNA-DNA hybrid that forms in the active site is maintained, and the newly synthesized RNA strand separates from the DNA template strand and emerges through the RNA exit channel. The RNA polymerase is capable of synthesizing RNA at a rate of 30 to 100 nucleotides per second. However, it sometimes pauses and even slides backward (backtracks). This phenomenon often occurs when a helical domain, or hairpin, forms in the RNA as it exits the RNA exit channel, when the newly synthesized RNA contains inverted-repeated sequences. It is not clear why hairpins cause pausing and backtracking, but they may pull the RNA polymerase backward or bind to it and change its conformation. Backtracking creates special problems for the TEC. When the RNA polymerase is forced backward, it pushes the 3′ end of the newly synthesized RNA forward, driving it into the secondary channel through which the nucleotides enter, as shown in Figure 2.13. It would remain this way, permanently blocked, except for the action of two proteins called GreA and GreB. These proteins insert their N termini into the secondary channel and cleave the 3′ end of the RNA in the channel until it is in its proper place in the active site so that transcription can continue.

RNA polymerase pausing