Читать книгу Snyder and Champness Molecular Genetics of Bacteria - Tina M. Henkin - Страница 158

While factor-independent terminators are easily recognizable, the factor-dependent transcription terminators have very little sequence in common with each other and therefore are not readily apparent. The major termination factor in E. coli is called Rho (ρ). The ρ factor can be found in most types of bacteria, and this type of termination is likely to be widely conserved.

Any model for how the ρ factor terminates transcription at ρ-dependent termination sites has to incorporate the following facts. First, ρ usually causes the termination of RNA synthesis only if the RNA is not being translated. In bacteria, which lack a nuclear membrane, translation can begin on a nascent RNA before transcription is complete (see the introduction). Second, ρ is an RNA-dependent ATPase that cleaves ATP to get energy, but its ATPase activity is dependent on the presence of RNA. Finally, ρ is also an RNA-DNA helicase. It is similar to the DNA helicases that separate the strands of DNA during replication, but it unwinds only a double helix with RNA in one strand and DNA in the other.

Figure 2.15 illustrates a current model for how ρ terminates transcription. The ρ protein forms a hexameric (six-member) ring made up of six subunits encoded by the rho gene. This ring binds to a sequence in the RNA called the rut (for rho utilization) site. However, ρ can bind to a rut site in the RNA only if the RNA in this region is not occupied by ribosomes during translation of an mRNA; for example, ρ can bind if translation has terminated upstream at a termination codon (see below). The rut sites are not very distinctive but are about 40 nucleotides long and have many C’s and have minimal secondary structure in the mRNA. Once ρ has bound to a rut site through the outside of its ring, the RNA then passes through the central hole, and the ring then moves along the RNA in the 5′-to-3′ direction, following the RNA polymerase. Energy for this movement is provided by the cleavage of ATP to adenosine diphosphate (ADP) by the ATPase activity of ρ. The ρ factor ring rotates down the RNA behind the RNA polymerase at a speed of about 60 nucleotides per second. However, RNA polymerase is capable of transcribing at 100 nucleotides per second, so ρ factor can catch up only if the RNA polymerase pauses at a ρ-dependent termination site. Then, the ρ factor can catch up to the RNA polymerase, and its RNA-DNA helicase activity disrupts the RNA-DNA hybrid in the transcription bubble, stopping transcription and releasing the RNA polymerase from the DNA template. While this model accounts for most of the known activities of ρ, it leaves unanswered the question of how ρ can access the RNA-DNA helix, which is inside the RNA polymerase. One possibility is that the RNA polymerase partially opens up when it is paused at a ρ-dependent termination site. The coupling of transcription termination to translation blockage promotes termination of transcription when protein synthesis is interrupted. However, ρ-dependent termination is not very efficient, and transcription continues through a ρ-sensitive pause site as much as 50% of the time. ρ-dependent termination not only occurs at the ends of transcribed regions, but also accounts for ρ-dependent polarity (see “Polar Effects on Gene Expression” below).

Figure 2.11 Abortive transcription and RNA polymerase escape from the promoter. RNA polymerase can escape from the promoter only if more than 10 or 11 nucleotides are polymerized. At 12 nucleotides, the RNA transcript displaces the σ_3.2 region, which blocks the active-site channel. Abortive transcription results when short (<9 nucleotides) newly synthesized transcripts are released, and the complex returns to the open complex (RP_o) state. With RNA polymerase escape, σ is released, and transcription elongation can continue.

Figure 2.12 The transcription elongation complex (TEC). During elongation, nucleoside triphosphates (NTPs) enter through the secondary channel and are polymerized at the active site; the nascent RNA exits through the RNA exit channel. Modified from Geszvain K, Landick R, in Higgins NP (ed), The Bacterial Chromosome (ASM Press, Washington, DC, 2005).

Figure 2.13 Backtracked transcription elongation complex (TEC). Backward movement of RNA polymerase results in placement of the 3′ end of the nascent RNA within the secondary channel, which prevents entry of nucleoside triphosphate (NTP) substrates. GreA and GreB can enter the secondary channel to cleave the nascent RNA, which repositions the 3′ end of the transcript in the active site, allowing transcription elongation to continue.