The P-Site Loop of the Universally Conserved Bacterial Ribosomal Protein L5 Is Required for Maintaining Both Translation Rate and Fidelity.

The bacterial ribosomal 5S rRNA-binding protein L5 is universally conserved (uL5). It contains the so-called P-site loop (PSL), which contacts the P-site tRNA in the ribosome. Certain PSL mutations in yeast are lethal, suggesting that the loop plays an important role in translation. In this work, for the first time, a viable Escherichia coli strain was obtained with the deletion of the major part of the PSL (residues 73-80) of the uL5 protein. The deletion conferred cold sensitivity and drastically reduced the growth rate and overall protein synthesizing capacity of the mutant. Translation rate is decreased in mutant cells as compared to the control. At the same time, the deletion causes increased levels of -1 frameshifting and readthrough of all three stop codons. In general, the results show that the PSL of the uL5 is required for maintaining both the accuracy and rate of protein synthesis in vivo.

Robust regulation of transcription pausing in Escherichia coli by the ubiquitous elongation factor NusG.

Transcription elongation by multi-subunit RNA polymerases (RNAPs) is regulated by auxiliary factors in all organisms. NusG/Spt5 is the only universally conserved transcription elongation factor shared by all domains of life. NusG is a component of antitermination complexes controlling ribosomal RNA operons, an essential antipausing factor, and a transcription-translation coupling factor in Escherichia coli. We employed RNET-seq for genome-wide mapping of RNAP pause sites in wild-type and NusG-depleted cells. We demonstrate that NusG is a major antipausing factor that suppresses thousands of backtracked and nonbacktracked pauses across the E. coli genome. The NusG-suppressed pauses were enriched immediately downstream from the translation start codon but were also abundant elsewhere in open reading frames, small RNA genes, and antisense transcription units. This finding revealed a strong similarity of NusG to Spt5, which stimulates the elongation rate of many eukaryotic genes. We propose a model in which promoting forward translocation and/or stabilization of RNAP in the posttranslocation register by NusG results in suppression of pausing in E. coli.

A Cre Transcription Fidelity Reporter Identifies GreA as a Major RNA Proofreading Factor in Escherichia coli.

We made a coupled genetic reporter that detects rare transcription misincorporation errors to measure RNA polymerase transcription fidelity in Escherichia coli Using this reporter, we demonstrated in vivo that the transcript cleavage factor GreA, but not GreB, is essential for proofreading of a transcription error where a riboA has been misincorporated instead of a riboG. A greA mutant strain had more than a 100-fold increase in transcription errors relative to wild-type or a greB mutant. However, overexpression of GreB in ΔgreA cells reduced the misincorporation errors to wild-type levels, demonstrating that GreB at high concentration could substitute for GreA in RNA proofreading activity in vivo.

SuhB Associates with Nus Factors To Facilitate 30S Ribosome Biogenesis in Escherichia coli.

UNLABELLED: A complex of highly conserved proteins consisting of NusB, NusE, NusA, and NusG is required for robust expression of rRNA in Escherichia coli. This complex is proposed to prevent Rho-dependent transcription termination by a process known as "antitermination." The mechanism of this antitermination in rRNA is poorly understood but requires association of NusB and NusE with a specific RNA sequence in rRNA known as BoxA. Here, we identify a novel member of the rRNA antitermination machinery: the inositol monophosphatase SuhB. We show that SuhB associates with elongating RNA polymerase (RNAP) at rRNA in a NusB-dependent manner. Although we show that SuhB is required for BoxA-mediated antitermination in a reporter system, our data indicate that the major function of the NusB/E/A/G/SuhB complex is not to prevent Rho-dependent termination of rRNA but rather to promote correct rRNA maturation. This occurs through formation of a SuhB-mediated loop between NusB/E/BoxA and RNAP/NusA/G. Thus, we have reassigned the function of these proteins at rRNA and identified another key player in this complex. IMPORTANCE: As RNA polymerase transcribes the rRNA operons in E. coli, it complexes with a set of proteins called Nus that confer enhanced rates of transcription elongation, correct folding of rRNA, and rRNA assembly with ribosomal proteins to generate a fully functional ribosome. Four Nus proteins were previously known, NusA, NusB, NusE, and NusG; here, we discover and describe a fifth, SuhB, that is an essential component of this complex. We demonstrate that the main function of this SuhB-containing complex is not to prevent premature transcription termination within the rRNA operon, as had been long claimed, but to enable rRNA maturation and a functional ribosome fully competent for translation.

Visualizing translocation dynamics and nascent transcript errors in paused RNA polymerases in vivo.

BACKGROUND: Transcription elongation is frequently interrupted by pausing signals in DNA, with downstream effects on gene expression. Transcription errors also induce prolonged pausing, which can lead to a destabilized genome by interfering with DNA replication. Mechanisms of pausing associated with translocation blocks and misincorporation have been characterized in vitro, but not in vivo. RESULTS: We investigate the pausing pattern of RNA polymerase (RNAP) in Escherichia coli by a novel approach, combining native elongating transcript sequencing (NET-seq) with RNase footprinting of the transcripts (RNET-seq). We reveal that the G-dC base pair at the 5' end of the RNA-DNA hybrid interferes with RNAP translocation. The distance between the 5' G-dC base pair and the 3' end of RNA fluctuates over a three-nucleotide width. Thus, the G-dC base pair can induce pausing in post-translocated, pre-translocated, and backtracked states of RNAP. Additionally, a CpG sequence of the template DNA strand spanning the active site of RNAP inhibits elongation and induces G-to-A errors, which leads to backtracking of RNAP. Gre factors efficiently proofread the errors and rescue the backtracked complexes. We also find that pausing events are enriched in the 5' untranslated region and antisense transcription of mRNA genes and are reduced in rRNA genes. CONCLUSIONS: In E. coli, robust transcriptional pausing involves RNAP interaction with G-dC at the upstream end of the RNA-DNA hybrid, which interferes with translocation. CpG DNA sequences induce transcriptional pausing and G-to-A errors.

Studying the properties of domain I of the ribosomal protein l1: incorporation into ribosome and regulation of the l1 operon expression.

L1 is a conserved protein of the large ribosomal subunit. This protein binds strongly to the specific region of the high molecular weight rRNA of the large ribosomal subunit, thus forming a conserved flexible structural element--the L1 stalk. L1 protein also regulates translation of the operon that comprises its own gene. Crystallographic data suggest that L1 interacts with RNA mainly by means of its domain I. We show here for the first time that the isolated domain I of the bacterial protein L1 of Thermus thermophilus and Escherichia coli is able to incorporate in vivo into the E. coli ribosome. Furthermore, domain I of T. thermophilus L1 can regulate expression of the L1 gene operon of Archaea in the coupled transcription-translation system in vitro, as well as the intact protein. We have identified the structural elements of domain I of the L1 protein that may be responsible for its regulatory properties.

Recombineering: using drug cassettes to knock out genes in vivo.

A 'gene knockout' or 'knockout' is a mutation that inactivates a gene function. These mutations are very useful for classical genetic studies as well as for modern techniques including functional genomics. In the past, knockouts of bacterial genes were often made by transposon mutagenesis. In this case, laborious screens are required to find a knockout in the gene of interest. Knockouts of other organisms have traditionally been made by first using in vitro genetic engineering to modify genes contained on plasmids or bacterial artificial chromosomes (BACs) and later moving these modified constructs to the organism of interest by cell culture techniques. Other methods utilizing a combination of genetic engineering and in vivo homologous recombination were inefficient at best. Recombineering provides a new way to generate knockout mutations directly on the bacterial chromosome or to modify any plasmid or BAC in vivo as a prelude to making knockouts in other organisms. The constructs are designed to the base pair and are not dependent on suitable restriction sites. A drug cassette can be placed anywhere within a gene or the open reading frame of the gene can be replaced with the drug cassette. Either way, the desired construct is selected for.

Recombineering: highly efficient in vivo genetic engineering using single-strand oligos.

Recombineering provides the ability to make rapid, precise, and inexpensive genetic alterations to any DNA sequence, either in the chromosome or cloned onto a vector that replicates in E. coli (or other recombineering-proficient bacteria), and to do so in a highly efficient manner. Complicated genetic constructs that are impossible to make with in vitro genetic engineering can be created in days with recombineering. Recombineering with single-strand DNA (ssDNA) can be used to create single or multiple clustered point mutations, small or large (up to 10kb) deletions, and small (10-20 base) insertions such as sequence tags. Using optimized conditions, point mutations can be made with such high frequencies that they can be found without selection. This technology excels at creating both directed and random mutations.

Nus transcription elongation factors and RNase III modulate small ribosome subunit biogenesis in Escherichia coli.

Escherichia coli NusA and NusB proteins bind specific sites, such as those in the leader and spacer sequences that flank the 16S region of the ribosomal RNA transcript, forming a complex with RNA polymerase that suppresses Rho-dependent transcription termination. Although antitermination has long been the accepted role for Nus factors in rRNA synthesis, we propose that another major role for the Nus-modified transcription complex in rrn operons is as an RNA chaperone insuring co-ordination of 16S rRNA folding and RNase III processing that results in production of proper 30S ribosome subunits. This contrarian proposal is based on our studies of nusA and nusB cold-sensitive mutations that have altered translation and at low temperature accumulate 30S subunit precursors. Both phenotypes are suppressed by deletion of RNase III. We argue that these results are consistent with the idea that the nus mutations cause altered rRNA folding that leads to abnormal 30S subunits and slow translation. According to this idea, functional Nus proteins stabilize an RNA loop between their binding sites in the 5' RNA leader and on the transcribing RNA polymerase, providing a topological constraint on the RNA that aids normal rRNA folding and processing.

NusA interaction with the α subunit of E. coli RNA polymerase is via the UP element site and releases autoinhibition.

Elongating Escherichia coli RNAP is modulated by NusA protein. The C-terminal domain (CTD) of the RNAP α subunit (αCTD) interacts with the acidic CTD 2 (AR2) of NusA, releasing the autoinhibitory blockade of the NusA S1-KH1-KH2 motif and allowing NusA to bind nascent nut spacer RNA. We determined the solution conformation of the AR2:αCTD complex. The αCTD residues that interface with AR2 are identical to those that recognize UP promoter elements A nusA-ΔAR2 mutation does not affect UP-dependent rrnH transcription initiation in vivo. Instead, the mutation inhibits Rho-dependent transcription termination at phage λtR1, which lies adjacent to the λnutR sequence. The Rho-dependent λtimm terminator, which is not preceded by a λnut sequence, is fully functional. We propose that constitutive binding of NusA-ΔAR2 to λnutR occludes Rho. In addition, the mutation confers a dominant defect in exiting stationary phase.

Structural basis for RNA recognition by NusB and NusE in the initiation of transcription antitermination.

Processive transcription antitermination requires the assembly of the complete antitermination complex, which is initiated by the formation of the ternary NusB-NusE-BoxA RNA complex. We have elucidated the crystal structure of this complex, demonstrating that the BoxA RNA is composed of 8 nt that are recognized by the NusB-NusE heterodimer. Functional biologic and biophysical data support the structural observations and establish the relative significance of key protein-protein and protein-RNA interactions. Further crystallographic investigation of a NusB-NusE-dsRNA complex reveals a heretofore unobserved dsRNA binding site contiguous with the BoxA binding site. We propose that the observed dsRNA represents BoxB RNA, as both single-stranded BoxA and double-stranded BoxB components are present in the classical lambda antitermination site. Combining these data with known interactions amongst antitermination factors suggests a specific model for the assembly of the complete antitermination complex.

Probing cellular processes with oligo-mediated recombination and using the knowledge gained to optimize recombineering.

Recombination with single-strand DNA oligonucleotides (oligos) in Escherichia coli is an efficient and rapid way to modify replicons in vivo. The generation of nucleotide alteration by oligo recombination provides novel assays for studying cellular processes. Single-strand exonucleases inhibit oligo recombination, and recombination is increased by mutating all four known exonucleases. Increasing oligo concentration or adding nonspecific carrier oligo titrates out the exonucleases. In a model for oligo recombination, λ Beta protein anneals the oligo to complementary single-strand DNA at the replication fork. Mismatches are created, and the methyl-directed mismatch repair (MMR) system acts to eliminate the mismatches inhibiting recombination. Three ways to evade MMR through oligo design include, in addition to the desired change (1) a C·C mismatch 6 bp from that change; (2) four or more adjacent mismatches; or (3) mismatches at four or more consecutive wobble positions. The latter proves useful for making high-frequency changes that alter only the target amino acid sequence and even allows modification of essential genes. Efficient uptake of DNA is important for oligo-mediated recombination. Uptake of oligos or plasmids is dependent on media and is 10,000-fold reduced for cells grown in minimal versus rich medium. Genomewide engineering technologies utilizing recombineering will benefit from both optimized recombination frequencies and a greater understanding of how biological processes such as DNA replication and cell division impact recombinants formed at multiple chromosomal loci. Recombination events at multiple loci in individual cells are described here.

Oligonucleotide recombination in Gram-negative bacteria.

This report describes several key aspects of a novel form of RecA-independent homologous recombination. We found that synthetic single-stranded DNA oligonucleotides (oligos) introduced into bacteria by transformation can site-specifically recombine with bacterial chromosomes in the absence of any additional phage-encoded functions. Oligo recombination was tested in four genera of Gram-negative bacteria and in all cases evidence for recombination was apparent. The experiments presented here were designed with an eye towards learning to use oligo recombination in order to bootstrap identification and development of phage-encoded recombination systems for recombineering in a wide range of bacteria. The results show that oligo concentration and sequence have the greatest influence on recombination frequency, while oligo length was less important. Apart from the utility of oligo recombination, these findings also provide insights regarding the details of recombination mediated by phage-encoded functions. Establishing that oligos can recombine with bacterial genomes provides a link to similar observations of oligo recombination in archaea and eukaryotes suggesting the possibility that this process is evolutionary conserved.

Structural and functional analysis of the E. coli NusB-S10 transcription antitermination complex.

Protein S10 is a component of the 30S ribosomal subunit and participates together with NusB protein in processive transcription antitermination. The molecular mechanisms by which S10 can act as a translation or a transcription factor are not understood. We used complementation assays and recombineering to delineate regions of S10 dispensable for antitermination, and determined the crystal structure of a transcriptionally active NusB-S10 complex. In this complex, S10 adopts the same fold as in the 30S subunit and is blocked from simultaneous association with the ribosome. Mass spectrometric mapping of UV-induced crosslinks revealed that the NusB-S10 complex presents an intermolecular, composite, and contiguous binding surface for RNAs containing BoxA antitermination signals. Furthermore, S10 overproduction complemented a nusB null phenotype. These data demonstrate that S10 and NusB together form a BoxA-binding module, that NusB facilitates entry of S10 into the transcription machinery, and that S10 represents a central hub in processive antitermination.

The structure of the R184A mutant of the inositol monophosphatase encoded by suhB and implications for its functional interactions in Escherichia coli.

The Escherichia coli product of the suhB gene, SuhB, is an inositol monophosphatase (IMPase) that is best known as a suppressor of temperature-sensitive growth phenotypes in E. coli. To gain insights into these biological diverse effects, we determined the structure of the SuhB R184A mutant protein. The structure showed a dimer organization similar to other IMPases, but with an altered interface suggesting that the presence of Arg-184 in the wild-type protein could shift the monomer-dimer equilibrium toward monomer. In parallel, a gel shift assay showed that SuhB forms a tight complex with RNA polymerase (RNA pol) that inhibits the IMPase catalytic activity of SuhB. A variety of SuhB mutant proteins designed to stabilize the dimer interface did not show a clear correlation with the ability of a specific mutant protein to complement the DeltasuhB mutation when introduced extragenically despite being active IMPases. However, the loss of sensitivity to RNA pol binding, i.e. in G173V, R184I, and L96F/R184I, did correlate strongly with loss of complementation of DeltasuhB. Because residue 184 forms the core of the SuhB dimer, it is likely that the interaction with RNA polymerase requires monomeric SuhB. The exposure of specific residues facilitates the interaction of SuhB with RNA pol (or another target with a similar binding surface) and it is this heterodimer formation that is critical to the ability of SuhB to rescue temperature-sensitive phenotypes in E. coli.

Recombineering: in vivo genetic engineering in E. coli, S. enterica, and beyond.

"Recombineering," in vivo genetic engineering with short DNA homologies, is changing how constructs are made. The methods are simple, precise, efficient, rapid, and inexpensive. Complicated genetic constructs that can be difficult or even impossible to make with in vitro genetic engineering can be created in days with recombineering. DNA molecules that are too large to manipulate with classical techniques are amenable to recombineering. This technology utilizes the phage lambda homologous recombination functions, proteins that can efficiently catalyze recombination between short homologies. Recombineering can be accomplished with linear PCR products or even single-stranded oligos. In this chapter we discuss methods of and ways to use recombineering.

Transcription antitermination by translation initiation factor IF1.

Bacterial translation initiation factor IF1 is an S1 domain protein that belongs to the oligomer binding (OB) fold proteins. Cold shock domain (CSD)-containing proteins such as CspA (the major cold shock protein of Escherichia coli) and its homologues also belong to the OB fold protein family. The striking structural similarity between IF1 and CspA homologues suggests a functional overlap between these proteins. Certain members of the CspA family of cold shock proteins act as nucleic acid chaperones: they melt secondary structures in nucleic acids and act as transcription antiterminators. This activity may help the cell to acclimatize to low temperatures, since cold-induced stabilization of secondary structures in nascent RNA can impede transcription elongation. Here we show that the E. coli translation initiation factor, IF1, also has RNA chaperone activity and acts as a transcription antiterminator in vivo and in vitro. We further show that the RNA chaperone activity of IF1, although critical for transcription antitermination, is not essential for its role in supporting cell growth, which presumably functions in translation. The results thus indicate that IF1 may participate in transcription regulation and that cross talk and/or functional overlap may exist between the Csp family proteins, known to be involved in transcription regulation at cold shock, and S1 domain proteins, known to function in translation.

Essentiality of ribosomal and transcription antitermination proteins analyzed by systematic gene replacement in Escherichia coli.

We describe here details of the method we used to identify and distinguish essential from nonessential genes on the bacterial Escherichia coli chromosome. Three key features characterize our method: high-efficiency recombination, precise replacement of just the open reading frame of a chromosomal gene, and the presence of naturally occurring duplications within the bacterial genome. We targeted genes encoding functions critical for processes of transcription and translation. Proteins from three complexes were evaluated to determine if they were essential to the cell by deleting their individual genes. The transcription elongation Nus proteins and termination factor Rho, which are involved in rRNA antitermination, the ribosomal proteins of the small 30S ribosome subunit, and minor ribosome-associated proteins were analyzed. It was concluded that four of the five bacterial transcription antitermination proteins are essential, while all four of the minor ribosome-associated proteins examined (RMF, SRA, YfiA, and YhbH), unlike most ribosomal proteins, are dispensable. Interestingly, although most 30S ribosomal proteins were essential, the knockouts of six ribosomal protein genes, rpsF (S6), rpsI (S9), rpsM (S13), rpsO (S15), rpsQ (S17), and rpsT (S20), were viable.

Importance of the 5 S rRNA-binding ribosomal proteins for cell viability and translation in Escherichia coli.

A specific complex of 5 S rRNA and several ribosomal proteins is an integral part of ribosomes in all living organisms. Here we studied the importance of Escherichia coli genes rplE, rplR and rplY, encoding 5 S rRNA-binding ribosomal proteins L5, L18 and L25, respectively, for cell growth, viability and translation. Using recombineering to create gene replacements in the E. coli chromosome, it was shown that rplE and rplR are essential for cell viability, whereas cells deleted for rplY are viable, but grow noticeably slower than the parental strain. The slow growth of these L25-defective cells can be stimulated by a plasmid expressing the rplY gene and also by a plasmid bearing the gene for homologous to L25 general stress protein CTC from Bacillus subtilis. The rplY mutant ribosomes are physically normal and contain all ribosomal proteins except L25. The ribosomes from L25-defective and parental cells translate in vitro at the same rate either poly(U) or natural mRNA. The difference observed was that the mutant ribosomes synthesized less natural polypeptide, compared to wild-type ribosomes both in vivo and in vitro. We speculate that the defect is at the ribosome recycling step.