T7 RNA polymerase studied by force measurements varying cofactor concentration.

RNA polymerases carry out the synthesis of an RNA copy from a DNA template. They move along DNA, incorporate nucleotide triphosphate (NTP) at the end of the growing RNA chain, and consume chemical energy. In a single-molecule assay using the T7 RNA polymerase, we study how a mechanical force opposing the forward motion of the enzyme along DNA affects the translocation rate. We also study the influence of nucleotide and magnesium concentration on this process. The experiment shows that the opposing mechanical force is a competitive inhibitor of nucleotide binding. Also, the single-molecule data suggest that magnesium ions are involved in a step that does not depend on the external load force. These kinetic results associated with known biochemical and mutagenic data, along with the static information obtained from crystallographic structures, shape a very coherent view of the catalytic cycle of the enzyme: translocation does not take place upon NTP binding nor upon NTP cleavage, but rather occurs after PPi release and before the next nucleotide binding event. Furthermore, the energetic bias associated with the forward motion of the enzyme is close to kT and represents only a small fraction of the free energy of nucleotide incorporation and pyrophosphate hydrolysis.

On the mechanism of inhibition of phage T7 RNA polymerase by lac repressor.

We study here the effect on phage T7 RNA polymerase activity of lac repressor bound downstream of the T7 promoter. When repressor binds in vitro at an operator centered at +13 or +15 with respect to transcription start, it does not prevent initiation, though the transcript yield is reduced. However, the processivity of the polymerase is depressed and transcript extension is blocked at positions +4 and +6, respectively. These results indicate that repressor and polymerase do not simply exclude each other from the promoter. Rather, they would come into steric conflict and compete for establishment or retention of interactions with the same segment of DNA, without this leading to the immediate displacement of either polymerase or repressor. The resulting destabilization of the transcription complex would depress both initiation rate and enzyme processivity. In contrast to the above results, little reduction in runoff transcription is observed when operator is centered at +47. The decreased sensitivity of polymerase to repressor bound at +47 versus +13 or +15 is likely to be due to the higher stability of the elongation complex during the transcription of downstream regions in comparison with the first transcribed nucleotides. We also show that under conditions of leaky repression and with operator centered at +13, a mutant T7 RNA polymerase showing normal promoter affinity but a slower elongation rate is more sensitive to repression than the wild-type enzyme, both in vitro and in vivo. In vitro, this higher sensitivity is largely due to a reduced ability of the mutant to overcome the elongation block at position +4. The parallel between the in vitro and in vivo data suggests that in vivo the repressor also does not prevent polymerase from binding to promoter, but interferes with subsequent steps in initiation and transcript extension, in this case presumably largely extension beyond +4.

The low processivity of T7 RNA polymerase over the initially transcribed sequence can limit productive initiation in vivo.

In vitro, after binding to the promoter to form a catalytically active complex, RNA polymerases abortively cycle over the first transcribed nucleotides (initial transcribed sequence or ITS) before leaving the promoter. With the bacteriophage T7 enzyme, the extent of abortive transcription varies with the nature of the ITS and with the elongation speed of the polymerase. Here, we compare in vitro and in vivo the yield of long transcripts from T7 promoters, with two different ITSs, the T7 gene10 and the lactose operon ITSs, and two different T7 RNA polymerases, the wild-type and a 2.7-fold slower mutant (G645A). The use of non-cognate ITS and/or slow polymerase decreases the yield of long transcripts in vitro and in vivo in a parallel fashion, with low polymerase speed and non-cognate ITS acting synergistically. In vitro, this decrease is mirrored by an increase in the average number of abortive cycles the enzyme undergoes before leaving the promoter; specifically, with the G645A mutant, transcript release is favored at any ITS position, whereas with the lac ITS it is particularly frequent at positions five and six following the incorporation of uridine residues. Hence, the more abortive cycles per long transcript synthesis in vitro, the lower the yield of long transcripts in vitro or in vivo. We conclude that the duration of abortive cycling can limit long transcript synthesis in vivo, as in vitro. Under conditions where cycling is minimal (wild-type polymerase, gene10 ITS), T7 promoter drives the synthesis of three long transcripts per second at 37 degrees C in vivo, a figure higher than for any Escherichia coli promoter.

Culture conditions differentially affect the translation of individual Escherichia coli mRNAs.

Our aim is to investigate whether changes in growth conditions can differentially affect the initiation of translation from individual Escherichia coli mRNAs that are not subjected to specific translational control. As a model system, we have constructed a series of point-mutated lacZ genes which differ in their Shine-Dalgarno (SD) sequence, their initiator codon, or the secondary structure around these elements. Alterations in growth conditions produced large (up to 8-fold) changes in the relative expression from these genes, which, we argue, stem from changes in their relative efficiencies of translation initiation. In particular, compared to genes bearing mutations outside the SD or initiator codon, genes mutated in these elements experience a significant decrease in their expression when cells are grown in minimal rather than rich medium; at 42 degrees C rather than 37 degrees C; or under amino acid starvation. We discuss the mechanisms underlying these effects, and evocate their possible generality.

Interdependence of translation, transcription and mRNA degradation in the lacZ gene.

We have constructed a collection of Escherichia coli strains which differ by point mutations in the ribosome binding site (RBS) that drives the translation of the lacZ gene. These mutations affect the Shine-Dalgarno sequence or the initiation codon, or create secondary structures that sequester these elements, and result in a 200-fold variation in beta-galactosidase expression. Surprisingly, these variations of expression are paralleled by nearly equivalent changes in the lacZ mRNA level. The ratio of the beta-galactosidase expression to the mRNA level reflects the average spacing between translating ribosomes: hence, paradoxically, mutations that affect translation initiation do not correspondingly change this spacing. Further analysis of the mRNA level variations shows that they originate from two independent mechanisms. When beta-galactosidase expression exceeds a threshold corresponding roughly to one translation event per transcript, the variations in the efficiency of translation initiation affect largely the chemical and functional lifetimes of the mRNA. We further show that the rate-limiting step in the chemical decay process is an RNase E-dependent cleavage, which is outcompeted by translation initiation. Below this expression threshold, the mRNA lifetime levels out and strain-to-strain variations in mRNA level arise solely from polarity effects. We suggest that, in this activity range, most mRNA molecules that escape polarity are crossed by a single ribosome, and hence are identical from the viewpoint of degradation. Altogether, the tight couplings between translation initiation on one hand, polarity and/or mRNA degradation on the other, result in translation initiation events being closely spaced in time even from inefficient RBS, at the expense of the mRNA level. Finally, we evocate the possible beneficial consequences of a coupling between translation, transcription and mRNA degradation, for the management of cellular resources.

Bacteriophage T7 RNA polymerase travels far ahead of ribosomes in vivo.

We show that in Escherichia coli at 32 degrees C, the T7 RNA polymerase travels over the lacZ gene about eightfold faster than ribosomes travel over the corresponding mRNA. We discuss how the T7 phage might exploit this high rate in its growth optimization strategy and how it obviates the possible drawbacks of uncoupling transcription from translation.

In the Escherichia coli lacZ gene the spacing between the translating ribosomes is insensitive to the efficiency of translation initiation.

We have constructed a series of 44 Escherichia coli strains in which the chromosomal region corresponding to the Ribosome Binding Site (RBS) of the lacZ gene, has been replaced by small DNA fragments harboring either RBSs from other genes, or artificial RBSs. The beta-galactosidase expression from these strains ranges from 1 to 130 per cent of that of the parental strain. Using this collection, we demonstrate here that strain-to-strain variations in expression are paralleled by nearly equivalent variations in lacZ mRNA content. We propose that, in this system, polarity and mRNA stability are tightly coupled to translation initiation, so that changes in RBS efficiency are detected mainly as changes in mRNA concentration rather than in the spacing between translating ribosomes. In addition, we show that the mRNA sequence immediately downstream from the initiator codon influences per se the lifetime of the lacZ mRNA. We discuss the mechanism of the interdependence between translation, transcription and degradation in this gene, and speculate about the general role of this interdependence in determining the expression of bacterial genes.