Transcription pause

R. J. Davenport, G. J. Wuite, R. Landick, and C. Bustamante, Single-molecule smdy of transcriptional pausing and arrest by E. coli RNA polymerase. Science 287, 2497-2500 (2000). 

Gong E, Yanofsky C. A transcriptional pause synchronizes translation with transcription in the tryptophanase operon leader region. J. Bacteriol. 2003 185 6472-6476. 

Backtracking of Pol II during the elongation phase can lead to transcriptional pausing and arrest. Pausing and arrest are blocks to transcription that... 

The lacZ contains a series of latent transcription terminators that are responsible for the polar effects of certain mutations. The terminators are in the vicinity of five positions 180,220,379,421, and 463 bp downstream of the transcription start point (67). No strong terminators are found further downstream from this region. Termination at all but the 421 position is p dependent in vitro, with efficiencies ranging from 8 to 56%. Those stop points correspond to 5 of the 11 transcriptional pause sites located within the region. 

Figure 37-6. The predominant bacterial transcription termination signal contains an inverted, hyphenated repeat (the two boxed areas) followed by a stretch of AT base pairs (top figure). The inverted repeat, when transcribed into RNA, can generate the secondary structure in the RNA transcript shown at the bottom of the figure. Formation of this RNA hairpin causes RNA polymerase to pause and subsequently the p termination factor interacts with the paused polymerase and somehow induces chain termination.

The most incisive studies of the problem at the molecular level are those from the Felsenfeld laboratory (see, for example. Refs. [75,76]). They have shown that at least under some circumstances, a polymerase can step around a nucleo-some, displacing it in cis, but not causing dissociation. It is not yet clear, however, if this mechanism is physiologically relevant and/or whether it is the only mechanism. There exist results in apparent conflict with this model (i.e.. Ref. [77]). That the in vivo process is certainly more complex than the in vitro models used to date is further indicated by the discovery of elongation factors that markedly increase transcriptional rates and suppress pausing (see, for example, Conaway and Conaway [78]). Thus, the question as to how transcription elongation occurs in a chromatin template remains at least partially unresolved. For a further discussion, see Jackson, this volume, p. 467. 

Clark, Studitsky, and Felsenfeld [88-92] have extensively evaluated transcription on single nucleosomes utilizing SP6 RNA polymerase. Based on their data they have developed a model which is shown in Fig. lA. This model, referred to as the spooling model, proposes that when transcription occurs into the first 25 bp of the nucleosome, the polymerase pauses (step 2). DNA behind the polymerase binds the octamer surface, which further inhibits the action of the polymerase due to steric hinderance of the polymerase s rotation (step 3). For transcription to continue the DNA behind the polymerase must dissociate to allow the polymerase to continue to transcribe an additional 35 bp (step 4). At this point sufficient surface of the octamer is now available to permit substantial levels of... 

Figure 2. (Left) Experimental setup in force flow measurements. Optical tweezers are used to trap beads but forces are applied on the RNApol-DNA molecular complex using the Stokes drag force acting on the left bead immersed in the flow. In this setup, force assists RNA transcription as the DNA tether between beads increases in length as a function of time, (a) The contour length of the DNA tether as a function of time and (b) the transcription rate as a function of the contour length. Pauses (temporary arrests of transcription) are shown as vertical arrows. (From Ref. 25.) (See color insert.)...

RNA but instead would have to start over. However, an encounter with certain DNA sequences results in a pause in RNA synthesis, and at some of these sequences transcription is terminated. The process of termination is not yet well understood in eukaryotes, so our focus is again on bacteria. E. coli has at least two classes of termination signals one class relies on a protein factor called p (rho) and the other is p-independent. 

Most p-independent terminators have two distinguishing features. The first is a region that produces an RNA transcript with self-complementary sequences, permitting the formation of a hairpin structure (see Fig. 8-2la) centered 15 to 20 nucleotides before the projected end of the RNA strand. The second feature is a highly conserved string of three A residues in the template strand that are transcribed into U residues near the 3 end of the hairpin. When a polymerase arrives at a termination site with this structure, it pauses (Fig. 26-7). Formation of the hairpin structure in the RNA disrupts several A=U base pairs in the RNA-DNA hybrid segment and may disrupt important interactions... 

FIGURE 26-7 Model for p-independent termination of transcription in f. coli. RNA polymerase pauses at a variety of DNA sequences, some of which are terminators. One of two outcomes is then possible the polymerase bypasses the site and continues on its way, or the complex undergoes a conformational change (isomerization). In the latter case, intramolecular pairing of complementary sequences in the newly formed RNA transcript may form a hairpin that disrupts the RNA-DNA hybrid and/or the interactions between the RNA and the polymerase, resulting in isomerization. An A=U hybrid region at the 3 end of the new transcript is relatively unstable, and the RNA dissociates completely, leading to termination and dissociation of the RNA molecule. This is the usual outcome at terminators. At other pause sites, the complex may escape after the isomerization step to continue RNA synthesis. 

The p-dependent terminators lack the sequence of repeated A residues in the template strand but usually include a CA-rich sequence called a rut (rho rhilization) element. The p protein associates with the RNA at specific binding sites and migrates in the 5 —>3 direction until it reaches the transcription complex that is paused at a termination site. Here it contributes to release of the RNA transcript. The p protein has an ATP-depend-ent RNA-DNA helicase activity that promotes translocation of the protein along the RNA, and ATP is hydrolyzed by p protein during the termination process. The detailed mechanism by which the protein promotes the release of the RNA transcript is not known. 

The function of all elongation factors is to suppress the pausing or arrest of transcription by the Pol II—TFIIF complex. 

When tryptophan levels are low, the ribosome pauses at the Trp codons in sequence 1. Formation of the paired structure between sequences 2 and 3 prevents attenuation, because sequence 3 is no longer available to form the attenuator structure with sequence 4. The 2 3 structure, unlike the 3 4 attenuator, does not prevent transcription. 

Although this picture seems clear and simple, many uncertainties remain. Transcription does not proceed evenly but by pauses and spurts. This has suggested the possibility of an "inchworm" type of movement of RNA polymerase.72-75 However, the observations may also be explained by variations in the sequence. There are both pausing or stalling sites76 and terminator sequences. The concentrations of the needed ribonucleotide triphosphate precursors will also affect the kinetics. In addition, defects in the... 

Termination of transcription involves stopping the elongation process at a region on the DNA template that signals termination and release of the RNA product and the RNA polymerase. Most terminators are similar in that they code for a double-stranded RNA stem-and-loop structure just preceding the 3 end of the transcript (fig. 28.11). Such structures cause RNA polymerase to pause, terminate, and detach. Two types of terminators have been distinguished. The first is sufficient without any accessory factors it contains about six uridine residues following the stem and loop (see fig. 28.11). The second type of terminator lacks the polyU stretch and requires a protein factor called rho to facilitate release. 

These results support the hypothesis that transcription read-through requires partial translation of the leader sequence. However, only if the translation pauses or stops in the region where the trp or arg codons occur is read-through... 

The function of the leader sequence is to fine tune expression of the trp operon based on the availability of tryptophan inside the cell. It does this as follows. The leader sequence contains four regions (Fig. 2, numbered 1-4) that can form a variety of base-paired stem-loop ( hairpin ) secondary structures. Now consider the two extreme situations the presence or absence of tryptophan. Attenuation depends on the fact that, in bacteria, ribosomes attach to mRNA as it is being synthesized and so translation starts even before transcription of the whole mRNA is complete. When tryptophan is abundant (Fig. 2a), ribosomes bind to the trp polycistronic mRNA that is being transcribed and begin to translate the leader sequence. Now, the two trp codons for the leader peptide lie within sequence 1, and the translational Stop codon (see Topic HI) lies between sequence 1 and 2. During translation, the ribosomes follow very closely behind the RNA polymerase and synthesize the leader peptide, with translation stopping eventually between sequences 1 and 2. At this point, the position of the ribosome prevents sequence 2 from interacting with sequence 3. Instead sequence 3 base-pairs with sequence 4 to form a 3 4 stem loop which acts as a transcription terminator. Therefore, when tryptophan is present, further transcription of the trp operon is prevented. If, however, tryptophan is in short supply (Fig. 2b), the ribosome will pause at the two trp codons contained within sequence 1. This leaves sequence 2 free to base pair with sequence 3 to form a 2 3 structure (also called the anti-terminator),... 

TrpR, which is a DNA binding repressor protein, regulates transcription initiation of the E. coli trpEDCBA operon. Under tryptophan limiting conditions, TrpR represses transcription initiation, whereas repression is relieved in the presence of excess tryptophan. Once transcription initiates the elongating transcription complex is subject to control by transcription attenuation (reviewed in References 5 and 6). The leader transcript can form three RNA secondary structures that are referred to as the pause hairpin, the antiterminator structure, and an intrinsic terminator hairpin. Because the antiterminator shares nucleotides in common with the terminator, their formation is mutually exclusive. The pause hairpin has two additional roles in this transcription attenuation mechanism it serves as an anti-antiterminator stmc-ture that prevents antiterminator formation, and it codes for a leader peptide. A model of the E. coli trp operon transcription attenuation mechanism is presented in Fig. 2a. 

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