Pseudoknot-dependent translational coupling in repBA genes of the IncB plasmid pMU720 involves reinitiation

Replication of the IncB miniplasmid pMU720 requires synthesis of the replication initiator protein, RepA, whose translation is coupled to that of a leader peptide, RepB. The unusual feature of this system is that translational coupling in repBA has to be activated by the formation of a pseudoknot immediately upstream of the repA Shine-Dalgarno sequence. A small antisense RNA, RNAI, controls replication of pMU720 by interacting with repBA mRNA to inhibit expression of repA both directly, by preventing formation of the pseudoknot, and indirectly, by inhibiting translation of repB. The mechanism of translational coupling in repBA was investigated using the specialized ribosome system, which directs a subpopulation of ribosomes that carry an altered anti-Shine-Dalgarno sequence to translate mRNA molecules whose Shine-Dalgarno sequences have been altered to be complementary to the mutant anti-Shine-Dalgarno sequence. Our data indicate that translation of repA involves reinitiation by the ribosome that has terminated translation of repB. The role of the pseudoknot in this process and its effect on the control of copy number in pMU720 are discussed.     

Transient idling of posttermination ribosomes ready to reinitiate protein synthesis

The fate of ribosomes between termination and initiation during protein synthesis is very basic, yet poorly understood. Here we found that translational reinitiation of the alkaline phosphatase gene occurs in Escherichia coli from an internal methionine codon when the authentic translation is prematurely terminated at a nonsense codon that is within seven codons upstream of the reinitiation codon (which we refer to as "reinitiation window"). Changing the reading frame downstream of the stop codon did not abolish the reinitiation, while inactivating the upstream initiation codon abolished the reinitiation. Moreover, depletion of the ribosome recycling factor (RRF), which disassembles posttermination ribosomes in conjunction with elongation factor G, did not influence the observed reinitiation. These findings suggest that posttermination ribosomes can undergo a transient idling state ready to reinitiate protein synthesis even in the absence of the Shine-Dalgarno (SD) sequence within the reinitiation window by evading disengagement from the mRNA.     

Computational design of orthogonal ribosomes

Orthogonal ribosomes (o-ribosomes), also known as specialized ribosomes, are able to selectively translate mRNA not recognized by host ribosomes. As a result, they are powerful tools for investigating translational regulation and probing ribosome structure. To date, efforts directed towards engineering o-ribosomes have involved random mutagenesis-based approaches. As an alternative, we present here a computational method for rationally designing o-ribosomes in bacteria. Working under the assumption that base-pair interactions between the 16S rRNA and mRNA serve as the primary mode for ribosome binding and translational initiation, the algorithm enumerates all possible extended recognition sequences for 16S rRNA and then chooses those candidates that: (i) have a similar binding strength to their target mRNA as the canonical, wild-type ribosome/mRNA pair; (ii) do not bind mRNA with the wild-type, canonical Shine-Dalgarno (SD) sequence and (iii) minimally interact with host mRNA irrespective of whether a recognizable SD sequence is present. In order to test the algorithm, we experimentally characterized a number of computationally designed o-ribosomes in Escherichia coli.     

Oxygen sensitivity of the nifLA promoter of Klebsiella pneumoniae.

Oxygen sensitivity of the nifLA promoter of Klebsiella pneumoniae has been demonstrated. Studies on the oxygen regulation of nifB-lacZ and nifH-lacZ fusions in the presence of the nifLA operon, which contains either an intact or a deleted nifL gene, indicate that possibly both the nifL promoter and the nifL product are responsible for nif repression by oxygen.     

Translation of the leaderless Caulobacter dnaX mRNA.

The expression of the Caulobacter crescentus homolog of dnaX, which in Escherichia coli encodes both the gamma and tau subunits of the DNA polymerase III holoenzyme, is subject to cell cycle control. We present evidence that the first amino acid in the predicted DnaX protein corresponds to the first codon in the mRNA transcribed from the dnaX promoter; thus, the ribosome must recognize the mRNA at a site downstream of the start codon in an unusual but not unprecedented fashion. Inserting four bases in front of the AUG at the 5' end of dnaX mRNA abolishes translation in the correct frame. The sequence upstream of the translational start site shows little homology to the canonical Shine-Dalgarno ribosome recognition sequence, but the region downstream of the start codon is complementary to a region of 16S rRNA implicated in downstream box recognition. The region downstream of the dnaX AUG, which is important for efficient translation, exhibits homology with the corresponding region from the Caulobacter hemE gene adjacent to the replication origin. The hemE gene also appears to be translated from a leaderless mRNA. Additionally, as was found for hemE, an upstream untranslated mRNA also extends into the dnaX coding sequence. We propose that translation of leaderless mRNAs may provide a mechanism by which the ribosome can distinguish between productive and nonproductive templates.     

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.     

Correlation between the effectiveness of translation initiation and secondary structure of mRNA in the hybrid gene cro-lacIZ

A set of plasmids containing short DNA deletions in the N-part of cro-lacIZ coding zone as constructed using Bal31 nuclease. Development of models of mRNA secondary structure was carried out stepwise beginning from the 5'-end by taking into consideration the hairpins first formed during mRNA synthesis. Comparison of the results of mRNA secondary structure determination and protein production analysis demonstrated a correlation between the efficiency of translation initiation and the appearance of a single-stranded region upon disruption of the mRNA local secondary structure in the translation initiation zone generated by ribosomes. These results confirm the suggestion of the central role played by the Shine-Dalgarno sequence in the generation of a single-stranded region. Some plasmids from the set are supposed to determine protein synthesis by a translation reinitiation mechanism in the absence of Shine--Dalgarno interaction. In this case, correlation between reinitiation efficiency and local disruption of the mRNA secondary structure by the terminating ribosome was also observed. The terminating ribosome that forms the single-stranded region near the initiation codon fulfils the major function of the Shine--Dalgarno interaction. In addition, the possible effect of another mRNA secondary structure region on the translation initiation efficiency is discussed.     

A Sequence Downstream of the Initiation Codon Is Essential for Cold Shock Induction of cspB of Escherichia coli

Cold shock induction of cspB has been shown to be primarily regulated at the mRNA level. Here, we demonstrate that the induction of cspB at low temperature also requires the translational cis-acting element called the downstream box (DB). Full induction of cspB at low temperature is achieved in the presence of both the Shine-Dalgarno sequence and DB. We propose that the DB sequence functions as a translational enhancer for the biosynthesis of CspB to bypass the inhibitory effect in translation caused by cold shock.     

The highly efficient translation initiation region from the Escherichia coli rpsA gene lacks a shine-dalgarno element

The translational initiation region (TIR) of the Escherichia coli rpsA gene, which encodes ribosomal protein S1, shows a number of unusual features. It extends far upstream (to position -91) of the initiator AUG, it lacks a canonical Shine-Dalgarno sequence (SD) element, and it can fold into three successive hairpins (I, II, and III) that are essential for high translational activity. Two conserved GGA trinucleotides, present in the loops of hairpins I and II, have been proposed to form a discontinuous SD. Here, we have tested this hypothesis with the "specialized ribosome" approach. Depending upon the constructs used, translation initiation was decreased three- to sevenfold upon changing the conserved GGA to CCU. However, although chemical probing showed that the mutated trinucleotides were accessible, no restoration was observed when the ribosome anti-SD was symmetrically changed from CCUCC to GGAGG. When the same change was introduced in the SD from a conventional TIR as a control, activity was stimulated. This result suggests that the GGA trinucleotides do not form a discontinuous SD. Others hypotheses that may account for their role are discussed. Curiously, we also find that, when expressed at moderate level (30 to 40% of total ribosomes), specialized ribosomes are only twofold disadvantaged over normal ribosomes for the translation of bulk cellular mRNAs. These findings suggest that, under these conditions, the SD-anti-SD interaction plays a significant but not essential role for the synthesis of bulk cellular proteins.     

Role of ribosome recycling factor (RRF) in translational coupling

RNA phage GA coat and lysis protein expression are translationally coupled through an overlapping termination and initiation codon UAAUG. Essential for this coupling are the proximity of the termination codon of the upstream coat gene to the initiation codon of the lysis gene (either a <3 nucleotide separation or physical closeness through a possible hairpin structure) but not the Shine-Dalgarno sequence. This suggests that the ribosomes completing the coat gene translation are exclusively responsible for translation of the lysis gene. Inactivation of ribosome recycling factor (RRF), which normally releases ribosomes at the termination codon, did not influence the expression of the reporter gene fused to the lysis gene. This suggests the possibility that RRF may not release ribosomes from the junction UAAUG. However, RRF is essential for correct ribosomal recognition of the AUG codon as the initiation site for the lysis gene.     

Evidence against an Interaction between the mRNA downstream box and 16S rRNA in translation initiation

Based on the complementarity of the initial coding region (downstream box [db]) of several bacterial and phage mRNAs to bases 1469 to 1483 in helix 44 of 16S rRNA (anti-downstream box [adb]), it has been proposed that db-adb base pairing enhances translation in a way that is similar to that of the Shine-Dalgarno (SD)/anti-Shine-Dalgarno (aSD) interaction. Computer modeling of helix 44 on the 30S subunit shows that the topography of the 30S ribosome does not allow a simultaneous db-adb interaction and placement of the initiation codon in the ribosomal P site. Thus, the db-adb interaction cannot substitute for the SD-aSD interaction in translation initiation. We have always argued that any contribution of the db-adb interaction should be most apparent on mRNAs devoid of an SD sequence. Here, we show that 30S ribosomes do not bind to leaderless mRNA in the absence of initiator tRNA, even when the initial coding region shows a 15-nucleotide complementarity (optimal fit) with the putative adb. In addition, an optimized db did not affect the translational efficiency of a leaderless lambda cI-lacZ reporter construct. Thus, the db-adb interaction can hardly serve as an initial recruitment signal for ribosomes. Moreover, we show that different leaderless mRNAs are translated in heterologous systems although the sequence of the putative adb's within helix 44 of the 30S subunits of the corresponding bacteria differ largely. Taken our data together with those of others (M. O'Connor, T. Asai, C. L. Squires, and A. E. Dahlberg, Proc. Natl. Acad. Sci. USA 96:8973-8978, 1999; A. La Teana, A. Brandi, M. O'Connor, S. Freddi, and C. L. Pon, RNA 6:1393-1402, 2000), we conclude that the db does not base pair with the adb.     

Autogenous translational regulation of the ribosomal MvaL1 operon in the archaebacterium Methanococcus vannielii.

The mechanisms for regulation of ribosomal gene expression have been characterized in eukaryotes and eubacteria, but not yet in archaebacteria. We have studied the regulation of the synthesis of ribosomal proteins MvaL1, MvaL10, and MvaL12, encoded by the MvaL1 operon of Methanococcus vannielii, a methanogenic archaebacterium. MvaL1, the homolog of the regulatory protein L1 encoded by the L11 operon of Escherichia coli, was shown to be an autoregulator of the MvaL1 operon. As in E. coli, regulation takes place at the level of translation. The target site for repression by MvaL1 was localized by site-directed mutagenesis to a region within the coding sequence of the MvaL1 gene commencing about 30 bases downstream of the ATG initiation codon. The MvaL1 binding site on the mRNA exhibits similarity in both primary sequence and secondary structure to the L1 regulatory target site of E. coli and to the putative binding site for MvaL1 on the 23S rRNA. In contrast to other regulatory systems, the putative MvaL1 binding site is located in a sequence of the mRNA which is not in direct contact with the ribosome as part of the initiation complex. Furthermore, the untranslated leader sequence is not involved in the regulation. Therefore, we suggest that a novel mechanism of translational feedback regulation exists in M. vannielii.