RNAP is an obligatory processive enzyme that must complete synthesis of the entire RNA chain since the transcripts that are released prematurely cannot re-enter transcription cycle. In bacteria, even in the absence of the tightly condensed chromatin, RNAP still encounters many roadblocks that either stall it temporarily or trigger RNA release. DNA-bound proteins, DNA lesions and various nucleic acid signals that induce pausing, arrest and termination (1
) can hinder RNAP progression along the template. Even at saturating substrate concentrations in vitro
, RNAP is moving in leaps, with its fast movement along the template punctuated by pauses (2
). Pausing plays numerous regulatory roles, is an obligatory step in termination pathways, and likely controls the overall rate of RNA chain elongation (3
RNAP is capable of making very long RNA chains (30 000 nt long in bacteria) but its rate is rather modest compared to DNA replicases: in Escherichia coli
, elongating RNAP (a complex of α2
ββ′ω subunits) moves at 20–90 nt/s (4
) whereas the replication fork advances 1000 nt/s (5
). This relatively inefficient operation of RNAP does not represent the limit of its catalytic potential since ‘fast’ substitutions in the β and β′ subunits that significantly increase its overall rate in vitro
have been described (6–10
). An attractive explanation rests on an assumption that the relatively slow rate of transcription is necessary for efficient regulation of gene expression where it provides for timely recruitment of, and response to regulatory factors, attenuation control, as well as determines folding pathways of the nascent RNA. Moreover, in bacteria transcription and translation are coupled, imposing additional restrictions on the speed that RNAP can attain without placing the nascent RNA in danger of release by Rho, which terminates the untranslated messages (4
). In other words, a catalytically perfect RNAP would leave little room for regulation and likely uncouple transcription and translation, while much slower RNAP would not be nimble enough to keep up with sustaining the RNA pool as it adapts to changing environmental and physiological conditions. Indeed, while different ‘fast’ and ‘slow’ viable alleles of RNAP have been isolated, they alter the apparent elongation rate in vitro
by less than 3- to 5-fold in each direction (7
), whereas mutations coding for much faster or slower enzyme variants are lethal (6
As substitutions that constitutively change the overall rate of RNA chain elongation appear to have a negative impact on fitness and are being removed by natural selection, the stage is set for transient alteration of RNAP kinetic properties by regulatory proteins. A subset of such factors (known as antiterminators) reduces pausing and termination (in other words, confers a fast phenotype) thereby helping RNAP transcribe long operons. These proteins use different nucleic acid targets during recruitment: λN binds the nascent RNA structure, λQ is recruited to the double-stranded DNA near the promoter, RfaH is recruited to the single-stranded non-template (NT) DNA strand during elongation (16–19
). The sites on RNAP to which these proteins bind are likely also distinct: we have recently concluded (20
) that RfaH binds to the β′-subunit clamp helices (β′ CH), whereas the target sites for λ regulators are still unknown but are thought to be quite different (21
). Yet all antiterminators share the ability to accelerate RNAP, suggesting that they induce similar changes in the transcription elongation complex (TEC).
To date, the changes that lead to the ‘antitermination’ modification of the RNAP have not been characterized in detail, and the molecular mechanism(s) by which elongation factors or substitutions in RNAP make the enzyme faster or slower is not known: they may control nucleotide addition at every template position by affecting the common rate-limiting step (which has not been elucidated for RNAP), or influence the TEC isomerization into off-pathway states at pause and termination sites (23
). We have proposed that at a pause site RNAP isomerizes into a state in which nucleotide addition is slowed due to transient changes in the active site architecture (), and from which different classes of pause and termination complexes arise (24
). We further speculated that substitutions in RNAP may alter its propensity towards the isomerization into the slow state. In a fast RNAP, the productive alignment of the 3′ RNA end in the active site, and consequently nucleotide addition, is favored. In contrast, a slow RNAP is more likely to lose the 3′end from the active site and enter a paused state, escape from which can be delayed by two orders of magnitude. Antiterminators may act in the same regulatory pathway, switching RNAP into the fast state. Slow, pause-prone enzymes should then be hypersensitive to modification by antiterminators, whereas fast RNAPs should appear resistant to further acceleration.
Figure 1. Unactivated intermediate mechanism. During rapid elongation (top horizontal arrows) RNAP active site (represented by two circles denoting the i and i−1 subsites) is optimized for facile catalysis: the 3′ OH of the nascent RNA is perfectly (more ...)
To test this hypothesis, we have determined effects of the E. coli
RfaH on RNA chain elongation by enzymes from an expanded panel of fast and slow RNAPs, including many previously uncharacterized kinetic variants. RfaH is recruited to the TEC at specific sites (called ops
) and is required for expression of several long operons (25
). Reports from several labs indicated that RfaH acts as an antiterminator both in vivo
and in vitro
), although the exact mechanism of its action remains elusive. RfaH structure and its binding site on the TEC are known (20
). RfaH increases the overall elongation rate in vitro
, reduces pausing at mechanistically distinct regulatory sites (known as class I and II, ), and facilitates bypass of some terminators (16
). Unlike other well-studied antiterminators (18
), RfaH does not require any accessory proteins (e.g. NusA or NusG), and its action is not dramatically affected by addition of the cellular extract. These features allow us to dissect RfaH effect on the TEC in a highly purified model system.
Our results indicate that RfaH acts primarily to reduce pausing: it fails to further accelerate the already fast mutants, as well as the wild-type RNAP transcribing under pause-free conditions, but is particularly effective with the enzymes that are prone to pausing because of amino acid changes or substrate deprivation. In contrast, RfaH cannot correct those slow phenotypes, which are due to the defects in the elementary catalytic steps rather than to the off-pathway events like pausing. We also show that the enzymes traditionally regarded as fast are better characterized as pause-resistant. We discuss our results within the framework of the current structural analysis of bacterial TECs.