Transcription elongation takes place after the recognition and binding of RNAP to a promoter. In this step, RNAP binds RNA nucleotides to each other in specific order that is complementary to the template DNA base order. Elongation cannot begin unless RNAP is completely clear of the promoter region. This escape is characterized by three different changes occurring to RNAP: dissociation of the recognition subunit ( factor) from RNAP, stabilization and robust binding of RNAP to the DNA template and primary movement of RNAP along the DNA template (Uptain et al., 1997).
Transcription elongation can be regulated by three different factors: signals, accessory factors and polymerase modifications. Signals that regulate transcription elongation
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involve growth conditions and stress responses. For example, the elongation of the rrn operon in E. coli is directly affected by its stringent response through the ppGpp signaling molecule. This molecule is responsible for the stringent response in E. coli that can lower the elongation rate of RNAP in E. coli and increase the frequency of rrnB operon premature termination (Kingston and Chamberlin, 1981). Similar to growth conditions, stress responses can affect transcription elongation as well. Vidovic and colleagues (2011)proteomically analyzed the wild-type E. coli 0157, which is adapted to cold, and the rpoS mutant strain and recognized a difference in the expression of 21 proteins between those two strains. They also found that the RpoS factor regulates the expression of several proteins when E. coli is exposed to a cold shock. Some of these proteins are important for adaptation to cold stress and others are vital for the normal central metabolic pathways of E. coli.
Accessory factors that regulate transcription elongation can be identified as proteins or small molecules that inhibit or promote transcription elongation and affect transcription elongation in different ways. Accessory factors can regulate transcription by affecting RNAP and/or its accessory proteins or the DNA itself. Some of these proteins, such as GreA and GreB, increase elongation rates by decreasing pausing or helping arrested transcription complexes resume elongation. Some other proteins affect the elongation process by binding to the nascent RNA transcript (such as the Rho protein described previously) or binding to the DNA template. Other protein factors affect the EC by altering its processivity through direct protein:protein interaction, leading to different changes in the activity of the EC. For instance, in phage , the N protein encoded by the phage initially binds to the nut site on the RNA strand. After that, a group of the host proteins (Nus factors and ribosomal S10 proteins) join the N protein on the nut site. This
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protein complex then interacts with the elongating RNAP and alters its protein components, leading to a more stable complex that is resistant to downstream pauses and termination signals (Friedman and Court, 1995; Uptain et al., 1997; Henkin, 2000). Bacterial RNAP consists of several subunits participating to achieve transcription. It has been found that these subunits have many sequence conservations among bacterial species (Allison et al., 1985; Archambault and Friesen, 1993). This leads to the suggestion that these proteins represent a possible target for transcription elongation regulation. Some of the RNAP subunits experience changes such as phosphorylation and glycosylation that can affect RNAP efficiency. However, in eukaryotes, these types of changes do not affect transcription unless they occur to large RNAP subunits. In eukaryotes, the C-terminal of the largest subunit of RNAP II contains a repeated sequence of heptapeptide amino acids, which are highly phosphorylated. RNAP II with a highly phosphorylated C-terminus has been found to be more efficient in elongation than RNAP II with a non-phosphorylated C-terminus (Uptain et al., 1997). In E. coli, phosphorylation of the and’ subunits was found to be associated with the shutoff of RNA synthesis. This connection emphasizes the association between phosphorylation and elongation regulation (Cozzone, 1988).
The transcription EC has the ability to introduce conformational changes to the transcribed DNA segment. As bacterial DNA is double-stranded and highly supercoiled, RNAP and its nascent RNA need to rotate around the DNA during elongation, which is not possible at all times as the DNA is stored in a highly compact structure and the RNAP itself is very large in comparison to the chromatin filaments. As a result, RNAP, with topoisomerase I ahead of it, forces the DNA to rotate around its axis, resulting in an increasing torsional stress in the downstream DNA. The introduced torsional stress may
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slow the movement of the proceeding elongation complex and can play an important role in transcription regulation (Liu and Wang, 1987; Kouzine and Levens, 2007; Kouzine et al., 2013). These facts led to the twin domain hypothesis by Liu and Wang in 1987. This hypothesis illustrates that ECs change the topology of DNA by leaving negative supercoiling behind them and increasing positive supercoiling ahead of them when their rotation is obstructed during active transcription (Liu and Wang, 1987). Moreover, the bacterial chromosome is structured in a way that is compatible with the bacterial cell cycle, which might play a role in elongation regulation. Bacterial chromosome consists of two main structures, which are the topologically constrained loops, or topological domains, and the higher structures known as macrodomains. The topologically constrained loops are formed by connections resulted from the negative supercoiling of the DNA. These loops are stabilized by the attachment of some DNA binding proteins along bacterial DNA to protect the cell against DNA over relaxation during replication and other processes by which the DNA is unwound (Postow et al., 2004). During the repair of DNA double-strand breaks, DNA topological domains help by keeping DNA ends close to each other (Wang et al., 2013). Macrodomains are suggested to be formed by some sequence specific binding proteins. The formation of these macrodomains is central for bacterial DNA compaction. There are four macrodomains in E. coli chromosome (Dame et al., 2011; Wang et al., 2013).