Translation at higher than an optimal level interferes with coupling at an intercistronic junction.

In pairs of adjacent genes co-transcribed on bacterial polycistronic mRNAs, translation of the first coding region frequently functions as a positive factor to couple translation to the distal coding region. Coupling efficiencies vary over a wide range, but synthesis of both gene products at similar levels is common. We report the results of characterizing an unusual gene pair, in which only about 1% of the translational activity from the upstream gene is transmitted to the distal gene. The inefficient coupling was unexpected because the upstream gene is highly translated, the distal initiation site has weak but intrinsic ability to bind ribosomes, and the AUG is only two nucleotides beyond the stop codon for the upstream gene. The genes are those in the filamentous phage IKe genome, which encode the abundant single-stranded DNA binding protein (gene V) and the minor coat protein that caps one tip of the phage (gene VII). Here, we have used chimeras between the related phage IKe and f1 sequences to localize the region responsible for inefficient coupling. It mapped upstream from the intercistronic region containing the gene V stop codon and the gene VII initiation site, indicating that low coupling efficiency is associated with gene V. The basis for inefficient coupling emerged when coupling efficiency was found to increase as gene V translation was decreased below the high wild-type level. This was achieved by lowering the rate of elongation and by decreasing the efficiency of suppression at an amber codon within the gene. Increasing the strength of the Shine-Dalgarno interaction with 16S rRNA at the gene VII start also increased coupling efficiency substantially. In this gene pair, upstream translation thus functions in an unprecedented way as a negative factor to limit downstream expression. We interpret the results as evidence that translation in excess of an optimal level in an upstream gene interferes with coupling in the intercistronic junction.

Emerging features of mRNA decay in bacteria.

The problem of mRNA decay in E. coli has recently seen exciting progress, with the discoveries that key degradation enzymes are associated together in a high molecular weight degradosome and that polyadenylation promotes decay. Recent advances make it clear that mRNA decay in bacteria is far more interesting enzymatically than might have been predicted. In-depth study of specific mRNAs has revealed multiple pathways for degradation. Which pathway a given mRNA follows appears to depend in large part on the location of the initiating endonucleolytic cleavage within the mRNA. During the steps of mRNA decay, stable RNA structures pose formidable barriers to the 3' --> 5' exonucleases. However, polyadenylation is now emerging as a process that plays an important role in maintaining the momentum of exonucleolytic degradation by adding single-stranded extensions to the 3' ends of mRNAs and their decay intermediates, thereby facilitating further exonuclease digestion.

Roles of polyadenylation and nucleolytic cleavage in the filamentous phage mRNA processing and decay pathways in Escherichia coli.

To define basic features of mRNA processing and decay in Escherichia coli, we have examined a set of mRNAs encoded by the filamentous phage f1 that have structures typical of bacterial mRNAs. They bear a stable hairpin stem-loop on the 3' end left from rho-independent termination and are known to undergo processing by RNase E. A small percentage of the f1 mRNAs were found to bear poly(A) tails that were attached to heterogeneous positions near the common 3' end. In a poly(A) polymerase-deficient host, the later-appearing processed mRNAs were stabilized, and a novel small RNA accumulated. This approximately 125-nt RNA proved to arise via RNase E cleavage from the 3'-terminal region of the mRNAs bearing the terminator. Normally ribosomes translating gene VIII appear to protect this cleavage site from RNase E, so that release of the fragment from the mRNAs occurs very slowly. The data presented define additional steps in the f1 mRNA processing and decay pathways and clarify how features of the pathways are used in establishing and maintaining the persistent filamentous phage infection. Although the primary mode of decay is endonucleolytic cleavage generating a characteristic 5' --> 3' wave of products, polyadenylation is involved in part in degradation of the processed mRNAs and is required for turnover of the 125-nt mRNA fragment. The results place polyadenylation at a later rather than an initiating step of decay. They also provide a clear illustration of how stably structured RNA 3' ends act as barriers to 3' --> 5' exonucleolytic mRNA decay.

Appropriate expression of filamentous phage f1 DNA replication genes II and X requires RNase E-dependent processing and separate mRNAs.

The products of in-frame overlapping genes II and X carried by the filamentous phage f1 genome are proteins with required but opposing functions in phage DNA replication. Their normal relative levels are important for continuous production of phage DNA without killing infected Escherichia coli hosts. Here we identify several factors responsible for determining the relative levels of pII and pX and that, if perturbed, alter the normal distribution of the phage DNA species in infected hosts. Translation of the two proteins is essentially relegated to separate mRNAs. The mRNAs encoding genes II and X are also differentially sensitive to cleavage dependent on rne, the gene encoding the only E. coli endo-RNase known to have a global role in mRNA stability. Whereas pII levels are limited at the level of mRNA stability, normal pX levels require transcription in sufficient amounts from the promoter for the smaller mRNA encoding only pX.

Translation limits synthesis of an assembly-initiating coat protein of filamentous phage IKe.

Translation is shown to be downregulated sharply between genes V and VII of IKe, a filamentous bacteriophage classed with the Ff group (phages f1, M13, and fd) but having only 55% DNA sequence identity to it. Genes V and VII encode the following proteins which are used in very different amounts: pV, used to coat the large number of viral DNA molecules prior to assembly, and pVII, used to serve as a cap with pIX in 3 to 5 copies on the end of the phage particle that emerges first from Escherichia coli. The genes are immediately adjacent to each other and are represented in the same amounts on the Ff and IKe mRNAs. Ff gene VII has an initiation site that lacks detectable intrinsic activity yet through coupling is translated at a level 10-fold lower than that of upstream gene V. The experiments reported reveal that by contrast, the IKe gene VII initiation site had detectable activity but was coupled only marginally to upstream translation. The IKe gene V and VII initiation sites both showed higher activities than the Ff sites, but the drop in translation at the IKe V-VII junction was unexpectedly severe, approximately 75-fold. As a result, gene VII is translated at similarly low levels in IKe- and Ff-infected hosts, suggesting that selection to limit its expression has occurred.

Filamentous phage IKe mRNAs conserve form and function despite divergence in regulatory elements.

As a means of determining whether there has been selection to conserve the basic pattern of filamentous phage mRNAs, the major mRNAs representing genes II to VIII have been defined for a phage distantly related to the Ff group specific for Escherichia coli hosts bearing F pili. Phage IKe has a genome with 55% identity with the Ff genome and infects E. coli strains bearing N pili. The results reveal a remarkably similar pattern of overlapping polycistronic mRNAs with a common 3' end and unique 5' ends. The IKe mRNAs, like the Ff phage mRNAs, represent a combination of primary transcripts and processed RNAs. However, examination of the sequences containing the RNA endpoint positions revealed that effectively the only highly conserved regulatory element is the rho-independent terminator that generates the common 3' end. Promoters and processing sites have not been maintained in identical positions, but frequently are placed so as to yield RNAs with similar coding function. By conserving the pattern of transcription and processing despite divergence in the regulatory elements and possibly the requirements for host, endoribonucleases, the results argue that the pattern is not simply fortuitous.

Functional analysis of filamentous phage f1 mRNA processing sites.

The abundant mRNAs used as templates for synthesis of filamentous phage f1 proteins are a combination of primary transcripts and 3' products of processing. The processing steps are mediated by host endoribonucleases. One of the enzymes implicated in f1 mRNA processing is RNase E, the only endonuclease thus far shown to have a global role in mRNA decay. By establishing the temperature-sensitive phenotypes of RNase E mutants and then inducing a transcription unit bearing cloned f1 processing sites, we show that RNase E is required for production of at least three of the processed RNAs. Using in vivo processing assays, we also test directly the regions implicated genetically in previous work to contain the processing sites. The sites function as discrete domains in a number of transcription units, show little influence of translation, but appear to have increased activity at the 5' terminus of an mRNA. From their functional properties, we suggest that the known processing sites from phage f1 that are dependent on RNase E may be representative of relatively late steps in rne-dependent cleavage pathways.

Mutational analysis of an inherently defective translation initiation site.

In a reverse of many studies of translational initiation sites, we have explored the basis for the inactivity of an apparently defective initiation site. Gene VII of the filamentous phage f1 has a translational start site with highly unusual functional properties and a sequence dissimilar to a prokaryotic ribosome binding site. The VII site shows no activity in assays of independent initiation, even in a deletion series designed to remove potentially interfering RNA secondary structure. Activity from the VII site is only observed if the site is coupled to a source of translation immediately upstream, but its efficiency is low at a one-nucleotide spacing from the stop codon of the upstream cistron and extremely sensitive to the distance between the stop codon and the gene VII AUG. These and other atypical characteristics of coupling distinguish the VII site from most coupled initiation sites. To identify the pattern of nucleotide substitutions that give the VII site the capacity for independent initiation, a series of designed and random point mutations were introduced in the sequence. Improving the Shine-Dalgarno complementarity from GG to GGAG or GGAGG made activity detectable, but at only low levels. Random substitutions, each increasing activity above background by a small increment, were found at 16 positions throughout the region of ribosome contact. These substitutions lengthened the Shine-Dalgarno complementarity or changed the G and C residues present in the wild-type site to A or T. Significant activity was not observed unless a strong Shine-Dalgarno sequence and a number of the up-mutations were present together. The nature and distribution of the substitutions and their agreement with the known preferences for nucleotides in initiation sites provide evidence that the VII site's major defect is its primary sequence overall. It appears to lack the specialized sequence required to bind free 30 S ribosomes, and thus depends on the translational coupling process to give it limited activity.

Characterization of elongating T7 and SP6 RNA polymerases and their response to a roadblock generated by a site-specific DNA binding protein.

As a means of generating homogeneous populations of elongation complexes with the RNA polymerases encoded by phages T7 and SP6, transcription has been carried out in vitro on templates associated with the Gln-111 mutant of EcoRI endonuclease. The Gln-111 protein, as a result of a single amino acid substitution at position 111, lacks cleavage function yet shows higher than wild-type affinity for the EcoRI recognition sequence GAATTC. On a series of linear and circular templates associated with Gln-111 protein, blockage of the phage RNA polymerase elongation complex is observed. The 3' endpoint of the major blocked-length RNA species, just 3 bp upstream from the GAATTC, reveals an extremely close approach of polymerase's leading edge to essential contacts between Gln-111 protein and its binding site. In contrast to E. coli RNA polymerase, which is blocked stably and quantitatively by Gln-111 protein (Pavco, P.A. and Steege, D. A. (1990) J. Biol. Chem. 265, 9960-9969), the phage polymerases show substantial levels of readthrough transcription beyond the protein block.

Distinctive patterns of translational reinitiation in the lac repressor mRNA: bridging of long distances by out-of-frame translation and RNA secondary structure, effects of primary sequence.

In the early region of the Escherichia coli lac repressor mRNA, translational reinitiation events triggered by nonsense codons occur over long distances and in a distinctive pattern not explained by simple use of the next available initiator triplet. Defined fusions of the restart sites to the lacZ coding region have been used to explore the basis for these reinitiation patterns and to ask whether the sites can function in independent initiation at the 5' end of an mRNA. The results obtained confirm earlier indications that the restart sites may have little or no inherent capacity for binding free 30S ribosomes. The data also add to growing evidence that primary sequence elements are important determinants of reinitiation efficiency. On the basis of the reinitiation activities for nonsense sites throughout the early region of the mRNA, we suggest that out-of-frame restarts and RNA secondary structure bridge long distances between the point of termination and downstream restart codons. Such bridging mechanisms could serve more generally as a means of propagating translational activity across long polycistronic mRNAs.

Phage fl mRNA processing in Escherichia coli: search for the upstream products of endonuclease cleavage, requirement for the product of the altered mRNA stability (ams) locus.

In Escherichia coli infected with the filamentous phage f1, a number of the polycistronic phage mRNA species are generated through post-transcriptional processing by host nuclease activity. In this paper we review experimental evidence assessing whether known RNases are involved in mediating these processing events, and we use S1 nuclease mapping methods to visualize putative upstream products of endonuclease cleavage. By examining f1 processing in a phage-infected host bearing a temperature-sensitive allele of the altered message stability locus (ams), we show that production of the major processed species requires a component of the host cell which functions in the messenger RNA decay process.

Elongation by Escherichia coli RNA polymerase is blocked in vitro by a site-specific DNA binding protein.

As a means of determining how elongating RNA polymerase responds to a protein in its path, transcription has been carried out in vitro with the purified Escherichia coli enzyme on templates associated with a sequence-specific DNA binding protein. The major RNA species generated is the length expected from RNA polymerase which has transcribed to the position of the bound protein and is unable to elongate further. The binding proteins used are two mutants of the EcoRI endonuclease which are defective in cleavage function but retain high affinity for the wild-type recognition sequence (Wright, D. J., King, K., and Modrich, P. (1989) J. Biol. Chem. 264, 11816-11821). Blockage of RNA polymerase occurs on linear and circular templates and, although efficient with both proteins, is more effective for the EcoRI derivative with the slower dissociation rate. The protein-blocked transcription complexes are stable over time and remain in an active form, resuming elongation when the blocking protein is displaced by an increase in ionic strength. These paused ternary complexes, if treated with the termination factor rho, undergo release. The 3' ends of the blocked-length RNAs from DNAs of distinct sequences reveal that the ternary complexes are positioned at a constant distance from the protein block, 14 nucleotides upstream of the EcoRI recognition sequence. This information is combined with exonuclease III footprinting data to position the 3' end of the nascent RNA chain in the ternary complex quite near (approximately 7 nucleotides) the leading edge of RNA polymerase.

Recognition and cleavage signals for mRNA processing lie within local domains of the phage f1 RNA precursors.

In Escherichia coli infected with the filamentous phage f1, a number of the abundant phage mRNAs, species C-G, are products of post-transcriptional processing. The approach of cloning the phage sequences likely to include the processing signals in a plasmid under transcriptional control of the lambda PL promoter (Blumer, K. J., and Steege, D. A. (1984) Nucleic Acids Res. 12, 1847-1861) was extended to additional sites to show that processing at all five major sites is mediated by nuclease activity encoded by the bacterial genome. Primer extension methods were used to map more accurately in the f1 DNA sequence the 5' end points of the processed RNAs. The DNA segments that encode the mRNA processing signals were delimited from parallel series of 5'-3' and 3'-5' deletions made into the regions in which the RNA 5' ends map. For each deletion variant, in vivo f1 mRNA processing activity was assessed by primer extension and S1 nuclease mapping methods. The data indicate that the processing signals are comprised of relatively local regions near the point of RNA cleavage. Whereas cleavage occurs at the 5' border of the sequences that comprise the D, E, and F processing sites and thereby places most of the recognition information in the mature or product portion of the precursor, it occurs more centrally within the region comprising the C site. The C and D sites function independently as substrates for cleavage, with the necessary information contained in regions of 70 and 90 nucleotides, respectively. Cleavage at the E site appears to require a region of 130 nucleotides which completely contains the F site. From the effects on processing activity of deleting sequences in this region, the overlapping E and F processing sites appear to consist functionally of two subdomains. Each has a cleavage site at its immediate 5' end which can be substituted by foreign sequences, but both utilize a common recognition domain downstream from the point of strand scission.

Translation of phage f1 gene VII occurs from an inherently defective initiation site made functional by coupling.

Expression of the filamentous phage f1 gene VII is shown to be translationally coupled to that of the upstream gene V. Fusions of the gene VII initiation site to the lacZ coding region were used to determine that initiation at the VII site is completely dependent on the process of translation having proceeded up to a stop codon immediately upstream from the VII site. Coupled expression from the VII site was found to be inefficient, proportional to the level of upstream translation, and very sensitive to the distance from the functional upstream stop codon. Independent expression from the VII site was not observed, even in a deletion series designed to remove potentially masking RNA structure. On the basis of the VII site's dissimilarity to ribosome binding site sequences and its properties overall, we suggest that it inherently lacks the features required for independent recognition by ribosomes, and acquires the ability to initiate synthesis of gene VII protein by virtue of the coupling process.

Bacteriophage lambda N gene leader RNA. RNA processing and translational initiation signals.

The positions of RNA processing events mediated by RNase III and of two ribosome binding sites have been defined in an in vitro transcript of 321 nucleotides initiated at the major leftward promoter (PL) of bacteriophage lambda. Purified RNase III makes two specific endonucleolytic cleavages in the transcript at points 88 and 197 nucleotides from the PL start. The positions of these cuts suggest a secondary structure for the RNase III recognition site which is similar to other RNase III sites in which double-stranded cleavage occurs. Structure mapping experiments reveal a pattern of cleavages made in the PL transcript by nucleases specific for single- or double-stranded RNA that support the structure proposed for the RNase III stem and provide clear evidence for the stem-loop formed in the RNA at the position of the N recognition (nutL) site. Our finding that ribosomes bind efficiently in vitro to the region of the PL transcript which includes the AUG codon at position 223 supports other evidence that this triplet is the N protein start codon. The existence of an additional ribosome binding site in the N gene leader region just downstream from the nutL site identifies a second position possibly used for translational initiation or regulation. Its occurrence suggests potential roles for ribosome interaction and/or translation of the leader RNA in regulating phage development and N gene expression.

Translational control of phage f1 gene expression by differential activities of the gene V, VII, IX and VIII initiation sites.

Phage-specific transcription and subsequent RNA processing in Escherichia coli infected with the filamentous phage (f1, M13, fd) generate a pool of abundant and relatively long-lived phage mRNA species encoding the four adjacent genes V, VII, IX and VIII. Yet the products of gene V and gene VIII are synthesized at much higher levels than the gene VII and gene IX proteins. To ask if the translational initiation sites heading these genes show corresponding differences in activity and/or functional properties, we have purified a number of the phage mRNAs from cells infected with f1 and examined them in in vitro initiation reactions. The ribosome binding patterns obtained for the phage mRNA species and for smaller defined RNA fragments containing selected initiator regions reveal a large range in apparent ribosome binding strengths. The gene V and gene VIII sites are recognized efficiently in each mRNA species in which they are present. Gene IX site activity appears to be limited by local mRNA structure: the site has undetectable or low ribosome binding activity in all of the phage mRNA species, but is at least tenfold more active if the RNA sequences required to form a potential hairpin stem-and-loop 15 nucleotides upstream from the initiator AUG have been removed. The gene VII site shows no evidence of interaction with ribosomes in any phage mRNA or RNA fragment tested. The same striking differences in initiation activity were observed in vivo by cloning small f1 DNA fragments containing gene V or gene VII initiation site sequences to drive beta-galactosidase synthesis. High levels of a gene V-beta-galactosidase fusion protein are initiated at the V site, but no detectable synthesis occurs from the VII site. If the VII site is preceded by all of the information encoding the upstream gene V, however, modest amounts of a fusion protein initiated at the VII site are produced. The overall results, in accord with the observed yields of proteins in the phage-infected cell, provide strong evidence that the properties of these translational initiation sites determine in a significant way the differential expression of phage f1 genes V, VII, IX and VIII.

lac repressor blocks in vivo transcription of lac control region DNA.

Transcription of the Escherichia coli lac repressor gene (lacI) in vivo produces monocistronic mRNAs with discrete 3' ends in the lac control region, although the DNA sequence of this region does not specify a strong termination signal of the traditional form. Direct analysis of lac transcripts was used to show that the DNA sequence alone does not provide the signal to end the repressor mRNA and to establish that of the proteins with specific binding sites on control region DNA only the lac repressor has a striking effect on the continuity of lacI gene transcription. RNAs with 3' ends in the control region sequence are major mRNA species produced from a repressor-bound template, reflecting as much as a 50-fold increase over their levels in the repressor's absence. Repressor binding to the operator thus has a dual function. In addition to blocking initiation of transcription from the lacZ promoter, repressor serves as a termination factor by setting the length of its own transcript and separating lacI and lacZYA into two distinct transcription units.

Messenger RNA conformation and ribosome selection of translational reinitiation sites in the lac repressor mRNA.

In the early region of the Escherichia coli lac repressor mRNA, the pattern of cleavage by nucleases specific for single or double-stranded RNA confirms the presence of secondary structures previously proposed to influence the pattern of translational reinitiation. These are positioned so as to mask a potential restart site centered on an in-phase GUG triplet corresponding to repressor amino acid position Val38. Our finding that a restart polypeptide initiated at the Val38 GUG codon is observed only in situations that that preclude base-pairing of adjacent mRNA sequences establishes a functional role for these structures in vivo. This evidence for structure, considered with the overall pattern of reinitiation events, suggests that local mRNA conformation is the major determinant that dictates ribosomal selection of restart sites within the early region of the repressor cistron.

Functional analysis of lac repressor restart sites in translational initiation and reinitiation.

To define some of the features that influence ribosomal recognition of translational restart sites in the lac repressor mRNA, recombinant DNA methods have been used to construct lacI-Z fusions in which lacZ gene expression is dependent upon initiation or reinitiation within lacI mRNA sequences. Reinitiation efficiencies, as assessed by beta-galactosidase levels in strains bearing such plasmids, appear to be determined by at least three features of the RNA between the termination codon and reinitiation codon: the presence of competing out-of-frame AUG or GUG triplets, the distance between termination and reinitiation points, and the extent to which restart sequences remain accessible to ribosomes. While some of the restart sites are used with substantial efficiency for reinitiation, they do not function detectably as primary initiators if placed at the 5' end of the lacZ mRNA. This finding concurs with our observation that relative to the wild-type initiator region, which is recovered in quantitative yield from in vitro initiation reactions, ribosome protection of the four restart sites occurs at more than 100-fold lower efficiencies. In part, the lack of initiation activity is rationalized by the striking potential these sequences have for forming stable secondary structures that sequester elements essential for ribosome binding. However, the differential functioning of the restart sites in primary initiation versus reinitiation must also reflect real differences in the mechanisms operative in the two events.

mRNA processing in Escherichia coli: an activity encoded by the host processes bacteriophage f1 mRNAs.

To examine the regions of the male-specific filamentous bacteriophage f1 genome that include signals for mRNA processing, the 5' endpoints of the major in vivo phage mRNAs have been located in the f1 DNA sequence by S1 nuclease mapping. The 5' ends of the purified mRNAs and additional phage-specific RNAs transiently visible early after infection occur in clusters of T-rich residues within genes that code for three phage proteins. When a 270-nucleotide region encompassing the 5' endpoints of three processed RNAs is transcribed as part of the bacteriophage lambda N mRNA in uninfected female cells, RNA 5' ends identical to ends of the three f1 RNAs are generated from the lambda-f1 precursor. This finding indicates that the mRNA processing activity is encoded by the bacterial host, and that its recognition sites are present in the local regions near the 5' ends which result from RNA cleavage. Several characteristics of f1 mRNA processing events have implications for the differential regulation of adjacent phage genes constrained in the same transcription unit, and may be representative of similar processing events occurring in the bacterial cell.