Termination and antitermination of mRNA synthesis in prokaryotes

The termination of mRNA chain in prokaryotes is brought about by certain termination signals on DNA. These DNA sequences providing termination sigrrai are called terminators. However, sometimes termination event, despite the presence of a terminator, can be prevented by some factor interacting with RNA polymerase. This phenomenon is called antitermination and the factor responsible for this phenomenon will be called antiterminator. It will be seen that the termination actually depends on the scrutiny of not only the DNA template, but also that of the product (RNA) obtained by transcription.

Rho (ρ) dependent and rho (ρ) independent termination of E. coli
The termination signals whenever found on DNA can be of two types : (i) those, which are recognized by a protein factor known as rho (ρ) factor, cause rho (ρ) dependent termination and (ii) those, which do not need rho (ρ) factor for termination cause rho (ρ) independent termination. Rho (ρ) factor is a protein of molecular weight 55,000 and was isolated from E. coli. It causes termination of most mRNA molecules in vitro at relatively low ionic strength, but is not needed for termination of some of the mRNAs. However, in vitro, high ionic strength needs to be maintained to demonstrate rho independent termination. In vivo, distinction between rho dependent and rho independent terminators was possible with the help of mutants deficient for rho factor, but still showing termination at the rho independent sites. In both cases (rho dependent and rho independent terminations), near the termination sites, there is similarity in secondary structure, which is generated, by palindromic sequences like the following :


In this palindrome the sequence on both strands is the same when read in 5' to 3' direction. In this palindrome,


also represents an inverse repeat giving dyad symmetry in the sequence. This causes complementarity between bases on same strand (GGT being complementary to ACC read in opposite directions), so that the latter can base pair to form a hairpin in mRNA or cruciform in DNA (Fig. 32.7).

Rho independent terminator differs from rho dependent terminator in the presence of a long run of U residues in mRNA (A residues in DNA) and of a G-C rich region in stem of hairpin structure formed by mRNA (A-T rich structure in DNA) (Fig. 32.8).

Palindromes in mRNA (which is being synthesized) cause formation of hairpins, which cause RNA polymerase to slow down or cause a pause in transcription. The events that follow this pause depend on the type of termination site, (i) At rho independent terminator, due to presence of a long run of U bases in mRNA and A bases in DNA template, there will be very weak rU-dA (RNA-DNA hybrid) base pairs needing very little energy to break. Since RNA polymerase slows down due to hairpin structure, rU-dA bond breaks at any one point causing release of mRNA. (ii) At fho-dependent terminator, rho protein is active as a tetramer. A model for its action proposes that rho (ρ) binds to the 5' end of nascent mRNA chain and moves along the length of mRNA. When RNA polymerase is having a pause due to hairpin structure, rho (ρ) catches up, interacts with RNA polymerase and releases the mRNA molecule (Fig. 32.9), from template accompanied with dissociation.of RNA polymerase and rho (ρ) factor.

Antitermination in phage.
In Regulation of Gene Expression 2.  Cricuit of Lytic Cycle and Lysogeny in Bacteriophages, we will show how early, middle and late genes are transcribed in a phage after infection, due to replacement of one sigma factor by another. In such cases new promoters will be needed for each set of genes which are expressed at different times and therefore, need to be recognized by new sigma factors. However, if early, middle and late genes are arranged in order and termination is prevented by an antiterminator, then after early genes, the middle genes and after middle genes, the late genes will be transcribed in a sequence without the need of new sigma factors (Fig. 32.10). This is what is achieved by the mechanism of antitermination in phage lambda (λ). There is an early gene N in phage lambda (λ)and its product pN is the antiterminator, which allows RNA polymerase to read past T_LI and T_R1 (termination sites on the left and right of the λDNA; the two DNA strands in λDNA are transcribed in opposite directions; see Fig. 32.11). The product of a delayed early gene Q (transcribed after early genes like middle genes) is pQ which similarly allows transcription of late genes without termination of mRNA at the end of delayed early genes. In this manner, the antitermination proteins like pN and pQ allow construction of a cascade for phage gene expression.

The protein pN needs a DNA site 'nut' ('N' utilization) before the termination site to indicate that pN should bind to RNA polymerase and help it to read through the termination site. In phage lambda, there is nutR on the right side and nutL on the left side; nutR lies between P_R (promoter on right) and T_R1 but close to T_R1 (terminator on right). Though, nutL also lies between P_L and T_L1, it lies very close to N gene and much before T_L1. At nut sites pN binds to RNA polymerase, which then continues transcribing past terminator, without caring for the T_R1 or T_L1 (Fig. 32.11).

There is yet another protein NusA (N utilization substance), which functions like rho (ρ), but is complementary to it. It displaces sigma after initiation and helps in termination (Fig. 32.4). The protein pN perhaps functions by displacing NusA, so that the termination may not take place. (For more details consult "Genes V" by Benjamin Lewin).