10. How does rho factor work?

9.10 How does rho factor work?

Key terms defined in this section
Polarity refers to the effect of a mutation in one gene in influencing the expression (at transcription or translation) of subsequent genes in the same transcription unit.

Rho factor is an essential protein in E. coli. It functions solely at the stage of termination. It is a ~275 kD hexamer of identical subunits. It functions as an ancillary factor for RNA polymerase; typically its maximum activity in vitro is displayed when it is present at ~10% of the concentration of the RNA polymerase (507).




Figure 9.27 A rho-dependent terminator has a sequence rich in C and poor in G preceding the actual site(s) of termination.

E. coli has relatively few rho-dependent terminators; most of the known rho-dependent terminators are found in phage genomes. The sequences required for rho-dependent termination are 50-90 bases long and lie upstream of the terminator. Their common feature is that the RNA is rich in C residues and poor in G residues. An example is given in Figure 9.27; C is by far the most common base (41%) and G is the least common base (14%). As a general rule the efficiency of a rho-dependent terminator increases with the length of the C-rich/G-poor region.




Figure 9.28 Rho factor pursues RNA polymerase along the RNA and can cause termination when it catches the enzyme pausing at a rho-dependent terminator.

Does rho factor act via recognizing DNA, RNA, or RNA polymerase? Rho has an ATPase activity, which is RNA-dependent and requires the presence of a polyribonucleotide, >50 or so bases long. This suggests that rho binds RNA. Probably an individual rho factor acts processively on a single RNA substrate. The "hot pursuit" model for rho action shown in Figure 9.28 supposes that it binds to a nascent RNA chain at some point upstream of the terminator. This might require a specific sequence or type of sequence, or attachment could occur just at a free 5′ end or some other exposed sequence. Rho then translocates along the mRNA.


How does rho catch up with RNA polymerase? One possibility is that rho simply moves along the transcript faster than RNA polymerase moves along the DNA. The enzyme pauses when it reaches a terminator, and termination occurs if rho catches it there.


Does rho release the transcript by acting directly on the DNA-RNA junction or indirectly by causing RNA polymerase to release RNA? In the absence of RNA polymerase, rho has a 5′-3′ helicase action that can cause an RNA-DNA hybrid to separate; hydrolysis of ATP is used to provide energy for the reaction.


These abilities suggest that rho can directly gain access to the stretch of RNA-DNA hybrid in the transcription bubble and cause it to unwind. Either as a result of this unwinding, or because of some interaction between rho and RNA polymerase, termination is completed by the release of rho and RNA polymerase from the nucleic acids (for review see 79).


The idea that rho moves along RNA leads to an important prediction about the relationship between transcription and translation. Rho must first have access to a binding sequence on RNA, and then must be able to move along the RNA. Either or both of these conditions may be prevented if ribosomes are translating an RNA. So the ability of rho factor to reach RNA polymerase at a terminator depends on what is happening in translation.


This model explains a puzzling phenomenon. In some cases, a nonsense mutation in one gene of a transcription unit prevents the expression of subsequent genes in the unit. This effect is called polarity. A common cause is the absence of the mRNA corresponding to the subsequent (distal) parts of the unit.




Figure 9.29 The action of rho factor may create a link between transcription and translation when a rho-dependent terminator lies soon after a nonsense mutation.

Suppose that there are rho-dependent terminators within the transcription unit, that is, before the terminator that usually is used. The consequences are illustrated in Figure 9.29. Normally these earlier terminators are not used, because the ribosomes prevent rho from reaching RNA polymerase. But a nonsense mutation releases the ribosomes, so that rho is free to attach to and/or move along the mRNA, enabling it to act on RNA polymerase at the terminator. As a result, the enzyme is released, and the distal regions of the transcription unit are never expressed. (Why should there be internal terminators? Perhaps they are simply sequences that by coincidence mimic the usual rho-dependent terminator.) Some stable RNAs that have extensive secondary structure are preserved from polar effects, presumably because the structure impedes rho attachment or movement.


rho mutations show wide variations in their influence on termination. The basic nature of the effect is a failure to terminate. But the magnitude of the failure, as seen in the percent of readthrough in vivo, depends on the particular target locus. Similarly, the need for rho factor in vitro is variable. Some (rho-dependent) terminators require relatively high concentrations of rho, while others function just as well at lower levels. This suggests that different terminators require different levels of rho factor for termination, and therefore respond differently to the residual levels of rho factor in the mutants (rho mutants are usually leaky).


Some rho mutations can be suppressed by mutations in other genes. This approach provides an excellent way to identify proteins that interact with rho. The β subunit of RNA polymerase is implicated by two types of mutation. First, mutations in the rpoB gene can reduce termination at a rho-dependent site. Second, mutations in rpoB can restore the ability to terminate transcription at rho-dependent sites in rho mutant bacteria.


Reviews
79: Das, A. (1993). Control of transcription termination by RNA-binding proteins. Ann. Rev. Biochem 62, 893-930.

Research
507: Roberts, J. W. (1969). Termination factor for RNA synthesis. Nature 224, 1168-1174.





Genes VII
Genes VII
ISBN: B000R0CSVM
EAN: N/A
Year: 2005
Pages: 382

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