During gene expression, information from a gene is used to synthesise a functional gene product, which can be often proteins. In non-protein coding genes, such as rRNA genes or tRNA genes, a functional RNA is produced. All known life (eukaryotes, prokaryotes and viruses) perform gene expression.
The steps in the gene expression are not always exactly the same. The process may be modified at the level of transcription, RNA splicing, translation, and post-translational modification of protein. Since control of the timing, amount of gene expression and location can have a deep effect on the functions of the gene in a cell or in a multicellular organism, it implies that gene regulation may contribute to evolutionary change. Gene expression is the most impotant level at which genotype gives rise to the phenotype in genetics.
Throughout this report, transcriptional anti-termination mechanism that regulates the termination of transcription is described, and emphasis is laid on how it can act as an important regulatory point in the control of prokaryotic and viral gene expression.
ANTITERMINATION PREVENTING TRANSCRIPTIONAL TERMINATION
During transcription, RNA polymerase moves along the DNA template, synthesising RNA, until terminator sequence is met. At this point, transcription stops, the completed product is released, and the enzyme dissociates from the DNA template by breaking all hydrogen bonds that hold the RNA-DNA hybrid. DNA duplex reforms. The termination event must not be simply regarded as a mechanism for generating the 3' end of the RNA molecule, but also as an opportunity to control gene expression. So the stages when RNA polymerase associates with DNA (initiation) or dissociates from it (termination) both are subject to specific control.
Bacteria utilise a variety of mechanisms to regulate transcription elongation and control gene expression in response to change in their environment. Among these, antitermination causes RNA polymerase to disregard a termination signal, thus continuing elongating its transcript until it reaches a second signal, an event called readthrough. Antitermination is controlled by an antiterminator protein when it attaches itself to the DNA near the commencement of the operon and then transfers to the RNA polymerase as it moves past towards the first termination signal. The termination signal is ignored by the enzyme due to the presence of the antiterminator protein, most probably by countering the destabilising properties of an intrinsic terminator or by avoiding stalling at a Rho-dependent terminator.
FIGURE 3 shows that antitermination can be used to control transcription by determining whether RNA polymerase terminates or reads through a particular terminator into the following region.
FIGURE 2 shows the antiterminator protein that attaches to the DNA and transfers to the RNA polymerase as it moves past, subsequently enabling the polymerase to continue transcription through termination signal number 1, so the second of the pair of genes in this operon is transcribed.
ANTITERMINATION IS A REGULATORY EVENT
The best characterised example of antitermination is provided by phage lambda, with which the phenomenon was discovered. There are two antitermination systems, one controlling early and the other controlling late viral gene expression.
Immediately after entering an Escherichia coli cell, transcription of the λ genome is initiated by the bacterial RNA polymerase attaching to two promoters, pL and pR, and transcribes two 'immediate-early' mRNAs, these terminating at positions tL1 and tR1:
The mRNA transcribed from pR to tR1 codes for a protein called Cro, one of the major regulatory proteins involved in the λ infection cycle. The second mRNA specifies the N protein, which is an antiterminator. The N protein attaches to the λ genome at sites nutL and nutR and transfers to the RNA polymerase as it passes. Now the RNA polymerase ignores the tL1 and tR1 terminators and continues transcription downstream of these points. The resulting mRNAs encode the 'delayed-early' proteins:
Early antitermination mediated by the λ N gene product and a short nucleotide sequence, nut (N utilisation), results in read-through of terminators so that essential downstream genes can be transcribed. The nut site has been subdivided into two motifs; a seven-nucleotide boxA sequence followed by a stem-loop boxB motif. The functional form of the λ nut site is thought to be the RNA rather than DNA. Four E. coli gene products are also required for N-mediated antitermination. These are the Nus (N utilization substance) proteins NusA, NusB, NusE, and NusG. Thus, the nut sequences in the transcribed RNA trigger modification of RNA polymerase by the λ N protein and E. coli Nus proteins such that transcription termination signals are no longer recognized.
The more simple late-gene antitermination system requires only the λ qut sequence, the λ Q protein, and the E. coli NusA protein for antitermination. The mechanism of antitermination in the E. coli ribosomal RNA (rrn) operons is unclear, but several specific features are shared in the rrn and A systems. The boxA and boxB elements, though reversed, are similar to those of λ. Mutations in either boxA or boxB decrease or eliminate rrn antitermination in vivo. Although wild-type boxB is required if both boxB and boxA are present, a short sequence containing only boxA is sufficient for antitermination activity. In addition, Nus factors appear to be involved in both systems. Although this involvement has not been investigated extensively in rrn antitermination, ribosomal RNA synthesis is defective in the nusBS mutant strain, suggesting a requirement for NusB.
Antitermination controlled by the N protein therefore ensures that the immediate-early and delayed-early proteins are synthesized at the appropriate times during the λ infection cycle. One of the delayed-early proteins, Q, is a second antiterminator that controls the switch to the late stage of the infection cycle.
Summary of the above regulatory event:
Some phages use antitermination to control progression from one stage of gene expression to the next. The antitermination protein (pN) coded by lambda gene N, is required for RNA polymerase to read through the terminators found at the ends of the immediate early genes. Later in phage infection, another antitermination protein, pQ is required. Both pN and pQ act on RNA polymerase as it passes nut and qut sites located at different relative positions in their respective transcription units. The pN protein recognises RNA polymerase carrying NusA when the enzyme passes the sequence boxB. The pN protein then binds to the complex and prevents termination by antagonising the action of NusA when the polymerase reaches the rho-dependent terminator.
