13. Summary
9.13 Summary |
A transcription unit comprises the DNA between a promoter, where transcription initiates, and a terminator, where it ends. One strand of the DNA in this region serves as a template for synthesis of a complementary strand of RNA. The RNA-DNA hybrid region is short and transient, as the transcription "bubble" moves along DNA. The RNA polymerase holoenzyme that synthesizes bacterial RNA can be separated into two components. Core enzyme is a multimer of structure α 2ββ′σ that is responsible for elongating the RNA chain. Sigma factor is a single subunit that is required at the stage of initiation for recognizing the promoter.
Core enzyme has a general affinity for DNA. The addition of sigma factor reduces the affinity of the enzyme for nonspecific binding to DNA, but increases its affinity for promoters. The rate at which RNA polymerase finds its promoters is too great to be accounted for by diffusion and random contacts with DNA; direct exchange of DNA sequences held by the enzyme may be involved.
Bacterial promoters are identified by two short conserved sequences centered at V35 and V10 relative to the startpoint. Most promoters have sequences that are well related to the consensus sequences at these sites. The distance separating the consensus sequences is 16-18 bp. RNA polymerase initially "touches down" at the V35 sequence and then extends its contacts over the V10 region. The enzyme covers ~77 bp of DNA. The initial "closed" binary complex is converted to an "open" binary complex by melting of a sequence of ~12 bp that extends from the V10 region to the startpoint. The A PT-rich base pair composition of the V10 sequence may be important for the melting reaction.
The binary complex is converted to a ternary complex by the incorporation of ribonucleotide precursors. There are multiple cycles of abortive initiation, during which RNA polymerase synthesizes and releases RNA chains of 2-9 bases without moving from the promoter. At the end of this stage, sigma factor is released, and the core enzyme contracts to cover ~50 bp. Then core enzyme moves along DNA, synthesizing RNA. A locally unwound region of DNA moves with the enzyme. The enzyme contracts further in size to cover only 30-40 bp when the nascent chain has reached 15-20 nucleotides; then it continues to the end of the transcription unit.
The "strength" of a promoter describes the frequency at which RNA polymerase initiates transcription; it is related to the closeness with which its V35 and V10 sequences conform to the ideal consensus sequences, but is influenced also by the sequences immediately downstream of the startpoint. Negative supercoiling increases the strength of certain promoters. Transcription generates positive supercoils ahead of RNA polymerase and leaves negative supercoils behind the enzyme.
The core enzyme can be directed to recognize promoters with different consensus sequences by alternative sigma factors. In E. coli, such sigma factors are activated by adverse conditions, such as heat shock or nitrogen starvation. B. subtilis contains a single major sigma factor with the same specificity as the E. coli sigma factor, but also contains a variety of minor sigma factors. Another series of factors is activated when sporulation is initiated; sporulation is regulated by two cascades in which sigma factor replacements occur in the forespore and mother cell. A similar mechanism for regulating transcription is also used by phage SPO1.
The geometry of RNA polymerase-promoter recognition is similar for holoenzymes containing all sigma factors (except σ54). Each sigma factor causes RNA polymerase to initiate transcription at a promoter that conforms to a particular consensus at V35 and V10. Direct contacts between sigma and DNA at these sites have been demonstrated for E. coli σ70. It is not clear how contacts made with sigma factor relate to contacts made by subunits of core enzyme. An unanswered question on the use of sigma factors concerns the nature of the mechanism that allows one sigma to replace another.
Bacterial RNA polymerase terminates transcription at two types of sites. Intrinsic terminators contain a G PC-rich hairpin followed by a run of U residues. They are recognized in vitro by core enzyme alone. Rho-dependent terminators require rho factor both in vitro and in vivo; they have a stretch of 50-90 nucleotides preceding the site of termination that is rich in C and poor in G residues. Rho factor is an essential protein that acts as an ancillary termination factor, which recognizes RNA and acts at sites where RNA polymerase has paused. The termination activity requires ATP hydrolysis.
The Nus factors increase the efficiency of rho-dependent termination, and provide the means by which antitermination factors act. The NusB-S10 dimer recognizes the boxA sequence in the elongating RNA; NusA joins subsequently. Factor NusA and the initiation factor sigma are mutually exclusive associates of core enzyme.
Antitermination is used by some phages to regulate progression from one stage of gene expression to the next and (less often) in bacteria. The lambda gene N codes for an antitermination protein (pN) that is necessary to allow RNA polymerase to read through the terminators located at the ends of the immediate early genes. Another antitermination protein, pQ, is required later in phage infection. pN and pQ act on RNA polymerase as it passes specific sites (nut and qut, respectively). These sites are located at different relative positions in their respective transcription units. pN recognizes RNA polymerase carrying NusA when the enzyme passes the sequence boxB. pN then binds to the complex and prevents termination when the polymerase reaches the rho-dependent terminator.
