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Versatile genome editing can be facilitated by the insertion of DNA sequences into specific locations. Current protocols involving clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins rely on low efficiency homology-directed repair or non-homologous end joining with modified double-stranded DNA oligonucleotides as donors. Our simple protocol eliminates the need for expensive equipment, chemical and enzymatic donor DNA modification or plasmid construction by using polyethylene glycol-calcium to deliver non-modified single-stranded DNA oligonucleotides and CRISPR-Cas9 ribonucleoprotein into protoplasts. Plants regenerated via edited protoplasts achieved targeted insertion frequencies of up to 50.0% in Nicotiana benthamiana and 13.6% in rapid cycling Brassica oleracea without antibiotic selection. Using a 60-nt donor containing 27 nt in each homologous arm, 6 of 22 regenerated N. benthamiana plants showed targeted insertions, and one contained a precise insertion of a 6-bp HindIII site. The inserted sequences were transmitted to the next generation and invite the possibility of future exploration of versatile genome editing by targeted DNA insertion in plants.

The Gram-negative bacterium Pseudomonas taiwanensis is a novel bacterium that uses shrimp shell waste as its sole sources of carbon and nitrogen. It is a versatile bacterium with potential for use in biological control, with activities including toxicity toward insects, fungi, and the rice pathogen Xanthomonas oryzae pv.oryzae (Xoo). In this study, the complete 5.08-Mb genome sequence of P. taiwanensis CMS was determined by a combination of NGS/Sanger sequencing and optical mapping. Comparison of optical maps of seven Pseudomonas species showed that P. taiwanensis is most closely related to P. putida KT 2400. We screened a total of 11,646 individual Tn5-transponson tagged strains to identify genes that are involved in the production and regulation of the iron-chelator pyoverdine in P. taiwanensis, which is a key anti-Xoo factor. Our results indicated that the two-component system (TCS) EnvZ/OmpR plays a positive regulatory role in the production of pyoverdine, whereas the sigma factor RpoS functions as a repressor. The knowledge of the molecular basis of the regulation of pyoverdine by P. taiwanensis provided herein will be useful for its development for use in biological control, including as an anti-Xoo agent.

Cellular responses to oxygen deprivation are essential for survival during energy crises in plants and animals. Hypoxia caused by submergence results in reprogramming of translation dynamic in plants, but the molecular mechanisms are not well understood. Here we show that Arabidopsis Snf1-related protein kinase 1 (SnRK1) phosphorylates the translation initiation factor eIFiso4G to regulate translation dynamic under submergence. In Arabidopsis, there are two eIFiso4G genes, eIFiso4G1 and eIFiso4G2, which belong to the eIF4G family. Both eIFiso4Gs were phosphorylated by SnRK1 under submergence. Interestingly, the eIFiso4G1 knockout mutant, but not the eIFiso4G2 mutant, became more sensitive to submergence, implying that eIFiso4G1 is involved in regulating submergence tolerance in Arabidopsis. Comparison of RNA sequences in the polysome fraction and the RNAs immunoprecipitated by eIFiso4G1 from Col-0 and the SnRK1 and eIFiso4G1 mutants revealed that lack of eIFiso4G1 phosphorylation disrupts the translation of specific mRNAs under submergence. Taken together, our findings suggest that the SnRK1-eIFiso4G1 relay controls the translation of an array of genes under hypoxia, including core hypoxia response genes and genes related to stress response and biosynthetic process.

Summary Plant protoplasts are useful for assessing the efficiency of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated protein 9 (Cas9) mutagenesis. We improved the process of protoplast isolation and transfection of several plant species. We also developed a method to isolate and regenerate single mutagenized Nicotianna tabacum protoplasts into mature plants. Following transfection of protoplasts with constructs encoding Cas9 and sgRNAs, target gene DNA could be amplified for further analysis to determine mutagenesis efficiency. We investigated N. tabacum protoplasts and derived regenerated plants for targeted mutagenesis of the phytoene desaturase (NtPDS) gene. Genotyping of albino regenerants indicated that all four NtPDS alleles were mutated in amphidiploid tobacco, and no Cas9 DNA could be detected in most regenerated plants.

Ethylene is an essential hormone in plants that is involved in low-oxygen and reoxygenation responses. As a key transcription factor in ethylene signaling, ETHYLENE INSENSITIVE3 (EIN3) activates targets that trigger various responses. However, most of these targets are still poorly characterized. Through analyses of our microarray data and the published Arabidopsis (Arabidopsis thaliana) EIN3 chromatin immunoprecipitation sequencing data set, we inferred the putative targets of EIN3 during anoxia-reoxygenation. Among them, GDH2, which encodes one subunit of glutamate dehydrogenase (GDH), was chosen for further studies for its role in tricarboxylic acid cycle replenishment. We demonstrated that both GDH1 and GDH2 are induced during anoxia and reoxygenation and that this induction is mediated via ethylene signaling. In addition, the results of enzymatic assays showed that the level of GDH during anoxia-reoxygenation decreased in the ethylene-insensitive mutants ein2-5 and ein3eil1 Global metabolite analysis indicated that the deamination activity of GDH might regenerate 2-oxoglutarate, which is a cosubstrate that facilitates the breakdown of alanine by alanine aminotransferase when reoxygenation occurs. Moreover, ineffective tricarboxylic acid cycle replenishment, disturbed carbohydrate metabolism, reduced phytosterol biosynthesis, and delayed energy regeneration were found in gdh1gdh2 and ethylene mutants during reoxygenation. Taken together, these data illustrate the essential role of EIN3-regulated GDH activity in metabolic adjustment during anoxia-reoxygenation.

Rice bacterial blight caused by Xanthomonas oryzae pv. oryzae (Xoo) is one of the most destructive rice diseases worldwide. Therefore, in addition to breeding disease-resistant rice cultivars, it is desirable to develop effective biocontrol agents against Xoo. Here, we report that a soil bacterium Pseudomonas taiwanensis displayed strong antagonistic activity against Xoo. Using matrix-assisted laser desorption/ionization imaging mass spectrometry, we identified an iron chelator, pyoverdine, secreted by P. taiwanensis that could inhibit the growth of Xoo. Through Tn5 mutagenesis of P. taiwanensis, we showed that mutations in genes that encode components of the type VI secretion system (T6SS) as well as biosynthesis and maturation of pyoverdine resulted in reduced toxicity against Xoo. Our results indicated that T6SS is involved in the secretion of endogenous pyoverdine. Mutations in T6SS component genes affected the secretion of mature pyoverdine from the periplasmic space into the extracellular medium after pyoverdine precursor is transferred to the periplasm by the inner membrane transporter PvdE. In addition, we also showed that other export systems, i.e., the PvdRT-OpmQ and MexAB-OprM efflux systems (for which there have been previous suggestions of involvement) and the type II secretion system (T2SS), are not involved in pyoverdine secretion.

Highlight To illustrate the hypoxia signal mediated by SnRK1.1, phosphoproteomics was used to identify not only the new SnRK1.1 putative targets related to submergence, but also to gain an insight into early hypoxia signalling events., SNF1 RELATED PROTEIN KINASE 1 (SnRK1) is proposed to be a central integrator of the plant stress and energy starvation signalling pathways. We observed that the Arabidopsis SnRK1.1 dominant negative mutant (SnRK1.1 K48M) had lower tolerance to submergence than the wild type, suggesting that SnRK1.1-dependent phosphorylation of target proteins is important in signalling pathways triggered by submergence. We conducted quantitative phosphoproteomics and found that the phosphorylation levels of 57 proteins increased and the levels of 27 proteins decreased in Col-0 within 0.5–3h of submergence. Among the 57 proteins with increased phosphorylation in Col-0, 38 did not show increased phosphorylation levels in SnRK1.1 K48M under submergence. These proteins are involved mainly in sugar and protein synthesis. In particular, the phosphorylation of MPK6, which is involved in regulating ROS responses under abiotic stresses, was disrupted in the SnRK1.1 K48M mutant. In addition, PTP1, a negative regulator of MPK6 activity that directly dephosphorylates MPK6, was also regulated by SnRK1.1. We also showed that energy conservation was disrupted in SnRK1.1 K48M, mpk6, and PTP1 S7AS8A under submergence. These results reveal insights into the function of SnRK1 and the downstream signalling factors related to submergence.

A novel dual-microbe Bacillus/yeast co-culture system is developed for cellulosic bioethanol production. A recombinant cellulosomal Bacillus subtilis that carries eight cellulosomal genes of Clostridium thermocellum, including one scaffolding protein gene (cipA), one cell-surface anchor gene (sdbA), two exo-glucosidase genes (celK and celS), two endoglucanase genes (celA and celR), and two xylanase genes (xynC and xynZ) was constructed. The partner microbes for the dual-microbe combination are the wild type kefir yeast Kluyveromyces marxianus KY3, K. marxianus KY3-NpaBGS, which carries a β-glucosidase (NpaBGS) gene from rumen fungus, and the K. marxianus KR5 strain, that harbors endoglucanase (egIII), exo-glucanase (cbhI) and NpaBGS genes. All three Bacillus/yeast co-culture systems could achieve the cellulose saccharification and ethanol conversion simultaneously better than KR5 alone. The combination of Bacillus/KY3-NpaBGS outperformed that of Bacillus/KY3, as they could produce β-glucosidase enzyme for the system. Although, KR5 produces two more kinds of cellulases than KY3-NpaBGS. Bacillus/KR5 could not perform better than Bacillus/KY3-NpaBGS. Our results suggest that the dual-microbe Bacillus/yeast co-culturing system could leverage the advantages from both microbes and have a great potential for integrating into consolidated bioprocessing system.

A number of APETALA2 (AP2)/ETHYLENE RESPONSE FACTOR (ERF) genes have been shown to function in abiotic and biotic stress responses, and these genes are often induced by multiple stresses. We report here the characterization of an AP2/ERF gene in Arabidopsis (Arabidopsis thaliana) that is specifically induced during hypoxia. We show that under normoxic conditions, the expression of AtERF73/HRE1 can be induced by exogenous addition of 1-aminocyclopropane-1-carboxylic acid and that a combination of hypoxia and 1-aminocyclopropane-1-carboxylic acid results in hyperinduction of AtERF73/HRE1 expression. In addition, hypoxic induction of AtERF73/HRE1 is reduced but not completely abolished in ethylene-insensitive mutants and in the presence of inhibitors of ethylene biosynthesis and responses. These results suggest that, in addition to ethylene, an ethylene-independent signal is also required to mediate hypoxic induction of AtERF73/HRE1. To assess the role of AtERF73/HRE1, we generated three independent RNA interference (RNAi) knockdown lines of AtERF73/HRE1. Under normoxic conditions, the AtERF73/HRE1-RNAi seedlings displayed increased ethylene sensitivity and exaggerated triple responses, indicating that AtERF73/HRE1 might play a negative regulatory role in modulating ethylene responses. Gas chromatography analyses showed that the production of ethylene was similar between wild-type and RNAi lines under hypoxia. Quantitative reverse transcription-polymerase chain reaction analyses showed that hypoxia-inducible genes could be affected by AtERF73/HRE1-RNAi lines in two different ways: hypoxic induction of glycolytic and fermentative genes was reduced, whereas induction of a number of peroxidase and cytochrome P450 genes was increased. Taken together, our results show that AtERF73/HRE1 is involved in modulating ethylene responses under both normoxia and hypoxia.

A mixed culture was constructed from compost of Napiergrass and sheep dung under anaerobic thermophilic conditions (60 °C). The native microflora was cultivated for numerous generations to obtain a stable mixed culture that can degrade lignocelluloses. The fifth generation of the mixed culture consisting of five main bacteria (Clostridium strain TCW1, Bacillus sp. THLA0409, Klebsiella pneumoniae THLB0409, Klebsiella oxytoca THLC0409, and Brevibacillus strain AHPC8120) was employed to investigate the effects of operating conditions on culture growth and production of biochemical products, including ethanol. The mixed culture effectively degraded a diverse range of lignocellulosic materials, including microcrystalline cellulose (avicel) and natural lignocelluloses (Napiergrass). Acetic acid, ethanol, and butanol were the main biochemical products produced by biological fermentation. Under optimal conditions, ethanol yields from avicel and Napiergrass reached maxima of 0.108 and 0.040 g g−1, representing ethanol productivities of 0.00055 and 0.00028 g g−1 h−1, respectively.

Understanding the function of detoxifying enzymes in plants toward xenobiotics is of major importance for phytoremediation applications. In this work, Arabidopsis (Arabidopsis thaliana; ecotype Columbia) seedlings were exposed to 0.6 mm acetochlor (AOC), 2 mm metolachlor (MOC), 0.6 mm 2,4,6-trinitrotoluene (TNT), and 0.3 mm hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX). In vivo glutathione (GSH) conjugation reactions of AOC, MOC, RDX, and TNT were studied in root cells using a multiphoton microscope. In situ labeling with monochlorobimane, used as a competitive compound for conjugation reactions with GSH, confirmed that AOC and MOC are conjugated in Arabidopsis cells. Reverse transcription-PCR established the expression profile of glutathione S-transferases (GSTs) and nitroreductases enzymes. Genes selected for this study were AtGSTF2, AtGSTU1, AtGSTU24, and two isoforms of 12-oxophytodienoate reductase (OPR1 and OPR2). The five transcripts tested were induced by all treatments, but RDX resulted in low induction. The mRNA level of AtGSTU24 showed substantial increase for all chemicals (23-fold induction for AOC, 18-fold for MOC, 5-fold for RDX, and 40-fold for TNT). It appears that GSTs are also involved in the conjugation reactions with metabolites of TNT, and to a lesser extent with RDX. Results indicate that OPR2 is involved in plant metabolism of TNT (11-fold induction), and in oxidative stress when exposed to AOC (7-fold), MOC (9-fold), and RDX (2-fold). This study comprises gene expression analysis of Arabidopsis exposed to RDX and AOC, which are considered significant environmental contaminants, and demonstrates the importance of microscopy methods for phytoremediation investigations.

The algal β-carotene hydroxylase gene Crchyb from Chlamydomonas reinhardtii, Czchyb from Chlorella zofingiensis, or Hpchyb from Haematococcus pluvialis and six other carotenoid-synthesis pathway genes were co-integrated into the genome of a yeast host. Each of these three algal genes showed a higher efficiency to convert β-carotene to downstream carotenoids than the fungal genes from Phaffia rhodozyma. Furthermore, the strain with Hpchyb displayed a higher carotenoid productivity than the strains integrated with Crchyb or Czchyb, indicating that Hpchyb is more efficient than Crchyb and Czchyb. These results suggest that β-carotene hydroxylase plays a crucial role in the biosynthesis of carotenoids.

Ethylene is known to play an essential role in mediating hypoxic responses in plants. Here, we show that in addition to regulating hypoxic responses, ethylene also regulates cellular responses in the reoxygenation stage after anoxic treatment in Arabidopsis. We found that expression of several ethylene biosynthetic genes and ethylene-responsive factors, including ERF1 and ERF2, was induced during reoxygenation. Compared with the wild type, two ethylene-insensitive mutants (ein2-5 and ein3eil1) were more sensitive to reoxygenation and displayed damaged phenotypes during reoxygenation. To characterize the role of ethylene, we applied microarray analysis to Col-0, ein2-5 and ein3eil1 under reoxygenation conditions. Our results showed that gene transcripts involved in reactive oxygen species (ROS) detoxification, dehydration response and metabolic processes were regulated during reoxygenation. Moreover, ethylene signalling may participate in regulating these responses and maintaining the homeostasis of different phytohormones. Our work presents evidence that ethylene has distinct functions in recovery after anoxia and provides insight into the reoxygenation signalling network.

Background As a strong fermentator, Saccharomyces cerevisiae has the potential to be an excellent host for ethanol production by consolidated bioprocessing. For this purpose, it is necessary to transform cellulose genes into the yeast genome because it contains no cellulose genes. However, heterologous protein expression in S. cerevisiae often suffers from hyper-glycosylation and/or poor secretion. Thus, there is a need to genetically engineer the yeast to reduce its glycosylation strength and to increase its secretion ability. Results Saccharomyces cerevisiae gene-knockout strains were screened for improved extracellular activity of a recombinant exocellulase (PCX) from the cellulose digesting fungus Phanerochaete chrysosporium. Knockout mutants of 47 glycosylation-related genes and 10 protein-trafficking-related genes were transformed with a PCX expression construct and screened for extracellular cellulase activity. Twelve of the screened mutants were found to have a more than 2-fold increase in extracellular PCX activity in comparison with the wild type. The extracellular PCX activities in the glycosylation-related mnn10 and pmt5 null mutants were, respectively, 6 and 4 times higher than that of the wild type; and the extracellular PCX activities in 9 protein-trafficking-related mutants, especially in the chc1, clc1 and vps21 null mutants, were at least 1.5 times higher than the parental strains. Site-directed mutagenesis studies further revealed that the degree of N-glycosylation also plays an important role in heterologous cellulase activity in S. cerevisiae. Conclusions Systematic screening of knockout mutants of glycosylation- and protein trafficking-associated genes in S. cerevisiae revealed that: (1) blocking Golgi-to-endosome transport may force S. cerevisiae to export cellulases; and (2) both over- and under-glycosylation may alter the enzyme activity of cellulases. This systematic gene-knockout screening approach may serve as a convenient means for increasing the extracellular activities of recombinant proteins expressed in S. cerevisiae.

Background Cellulose, which is the most abundant renewable biomass on earth, is a potential bio-resource of alternative energy. The hydrolysis of plant polysaccharides is catalyzed by microbial cellulases, including endo-β-1,4-glucanases, cellobiohydrolases, cellodextrinases, and β-glucosidases. Converting cellobiose by β-glucosidases is the key factor for reducing cellobiose inhibition and enhancing the efficiency of cellulolytic enzymes for cellulosic ethanol production. Results In this study, a cDNA encoding β-glucosidase was isolated from the buffalo rumen fungus Neocallimastix patriciarum W5 and is named NpaBGS. It has a length of 2,331 bp with an open reading frame coding for a protein of 776 amino acid residues, corresponding to a theoretical molecular mass of 85.1 kDa and isoelectric point of 4.4. Two GH3 catalytic domains were found at the N and C terminals of NpaBGS by sequence analysis. The cDNA was expressed in Pichia pastoris and after protein purification, the enzyme displayed a specific activity of 34.5 U/mg against cellobiose as the substrate. Enzymatic assays showed that NpaBGS was active on short cello-oligosaccharides from various substrates. A weak activity in carboxymethyl cellulose (CMC) digestion indicated that the enzyme might also have the function of an endoglucanase. The optimal activity was detected at 40°C and pH 5 ~ 6, showing that the enzyme prefers a weak acid condition. Moreover, its activity could be enhanced at 50°C by adding Mg2+ or Mn2+ ions. Interestingly, in simultaneous saccharification and fermentation (SSF) experiments using Saccharomyces cerevisiae BY4741 or Kluyveromyces marxianus KY3 as the fermentation yeast, NpaBGS showed advantages in cell growth, glucose production, and ethanol production over the commercial enzyme Novo 188. Moreover, we showed that the KY3 strain engineered with the NpaNGS gene can utilize 2 % dry napiergrass as the sole carbon source to produce 3.32 mg/ml ethanol when Celluclast 1.5 L was added to the SSF system. Conclusion Our characterizations of the novel β-glucosidase NpaBGS revealed that it has a preference of weak acidity for optimal yeast fermentation and an optimal temperature of ~40°C. Since NpaBGS performs better than Novo 188 under the living conditions of fermentation yeasts, it has the potential to be a suitable enzyme for SSF.

Ethylene plays an essential role in response to hypoxic stress in plants. In most plant species, 1-aminocyclopropane-1-carboxylate synthase (ACS) is the key enzyme that regulates the production of ethylene. We examined the expression of ACS genes in Arabidopsis during hypoxia. Our data showed that the expression of 4 of the 12 Arabidopsis ACS genes, ACS2, ACS6, ACS7, and ACS9, is induced during hypoxia with three distinct patterns. The hypoxic induction of ACS9 is inhibited by aminooxy acetic acid, an inhibitor of ethylene biosynthesis. In addition, the hypoxic induction of ACS9 is also reduced in etr1-1 and ein2-1, two ethylene insensitive mutants in ethylene-signaling pathways, whereas the addition of 1-aminocyclopropane-1-carboxylic acid, a direct precursor of ethylene, does not induce ACS9 under normoxic conditions. These results indicate that ethylene is needed, but not sufficient, for the induction of ACS9 during hypoxia. This pattern of regulation is similar to that of ADH that encodes alcohol dehydrogenase, which we have reported previously. In contrast, the increased ethylene production during hypoxia has an inhibitory effect on ACS2 induction in roots, whereas ethylene has no effect on the hypoxic induction of ACS6 and ACS7. Based on these results, we propose that two signaling pathways are triggered during hypoxia. One pathway leads to the activation of ACS2, ACS6, and ACS7, whereas the other pathway leads to the activation of ADH and ACS9.

Expression of the alcohol dehydrogenase gene (ADH) of Arabidopsis is induced during hypoxia. Because many plants increase their ethylene production in response to hypoxic stress, we examined in this report whether ethylene is involved in the hypoxic induction ofADH in Arabidopsis. We found that the hypoxic induction of ADH can be partially inhibited by aminooxy acetic acid, an inhibitor of ethylene biosynthesis. This partial inhibition can be reversed by the addition of 1-aminocyclopropane-1-carboxylic acid, a direct precursor of ethylene. In addition, the hypoxic induction of the ADH gene is also reduced inetr1-1 and ein2-1, two ethylene insensitive mutants in ethylene-signaling pathways, whereas the addition of exogenous ethylene or an increase in cellular ethylene alone does not induce ADH under normoxic conditions. Kinetic analyses of ADH mRNA accumulation indicated that an ethylene signal is required for the induction of ADHduring later stages of hypoxia. Therefore, we conclude that ethylene is needed, but not sufficient for, the induction of ADH in Arabidopsis during hypoxia.

We have adopted a hypoxic treatment system in which only roots were under hypoxic conditions. Through analyzing global transcriptional changes in both shoots and roots, we found that systemic signals may be transduced from roots to trigger responses in tissues not directly subjected to hypoxia. The molecular mechanisms of such systemic responses under flooding are currently largely unknown. Using ontological categorization for regulated genes, a systemic managing program of carbohydrate metabolism was observed, providing an example of how systemic responses might facilitate the survival of plants under flooding. Moreover, a proportion of gene expressions that regulated in shoots by flooding was affected in an ethylene signaling mutation, ein2-5. Many systemic-responsive genes involved in the systemic carbohydrate managing program, hormone responses and metabolism, ubiquitin-dependent protein degradation were also affected in ein2-5. These results suggested an important role of ethylene in mediation of hypoxic systemic responses. Genes associated with abscisic acid (ABA) biosynthesis are upregulated in shoots and down regulated in roots. An ABA signaling mutation, abi4-1, affects expression of several systemic responsive genes. These results suggested that regulation of ABA biosynthesis could be required for systemic responses. The implications of these results for the systemic responses of root-flooded Arabidopsis are discussed.

Abortive and productive initiation assays were used to study transcription initiation at the PRE promoter of phage lambda in vitro. Two parameters were measured: k2, the rate constant for the transition between closed and open complexes; and KB, the equilibrium constant for the initial binding of RNA polymerase to promoter DNA. In the absence of cII protein (which activates PRE) the PRE promoter was extremely weak as expected, with k2 = 4.0 X 10(-4) S-1 and KB = 1.0 X 10(7) M-1. The addition of cII protein resulted in about a 15-fold increase in KB and a 40-fold increase in k2. Thus, cII activation of PRE results both in enhanced binding of RNA polymerase to DNA to form closed complexes and in an enchanced rate of isomerization of closed to open complexes. In addition, we found that open complexes formed in the presence of cII protein were at least four times as stable as those formed in its absence. This suggests that RNA polymerase and cII protein may remain in close contact even after complexes are formed.

Abortive initiation and run-off transcription assays were used to study the effects of cy mutations on activation of the phage lambda PRE promoter by cII gene product. Six point mutations in the repeated T-T-G-C sequences that flank the -35 consensus region of PRE decreased the apparent affinity of the promoter for cII protein by factors of 4-16 relative to the wild-type affinity. Kinetic analyses of transcription initiation in the presence and absence of cII protein demonstrated that five of the six mutations did not significantly affect the intrinsic interaction of RNA polymerase with PRE. Thus, these mutations differ from other cy mutations, including those in the -35 consensus region, which affect the formation of polymerase-PRE closed complexes or the isomerization of closed complexes to open complexes but do not affect the binding of cII protein. A sixth T-T-G-C mutation, cy3001, may affect intrinsic initiation by RNA polymerase as well as cII binding.

The efficiency of translation of the cII gene of bacteriophage lambda is greatly reduced by the cII3059 mutation, a GUU----GAU (Val----Asp) change in the second cII codon. Mutations in the third and fourth codons of the cII gene, called ctr mutations, reverse this translation deficiency. Lambda cII3059 ctr-1, which has a GCA----ACA (Ala----Thr) change in the fourth cII codon, produces about half the normal level of cII activity in liquid cultures, and lambda cII3059 ctr-2 and lambda cII3059 ctr-3, which have identical CGT----CGC changes in the third codon, produce normal levels of cII activity in liquid culture. Since the cII protein of ctr-3 has the same primary sequence as that of lambda cII3059, the cII- phenotype of lambda cII3059 can be explained entirely by the deficiency of translating cII mRNA. We propose that ctr mutations increase translation efficiency by destabilizing a stable stem structure which can be formed by cII mRNA. The ctr mutations lie in an overlapping regulatory region which contains, in addition to sequence elements that influence the rate of cII translation, a region to which cII protein binds to activate transcription from the PRE promoter. The ctr-1 mutation alters the cII recognition sequence from 5'-T-T-G-C-N6T-T-G-C-3' to 5'-T-T-G-C-N6T-T-G-T-3', but has no effect on PRE activity. Since a C----T change in the first (5'-proximal) T-T-G-C sequence (to yield 5'-T-T-G-T-N6T-T-G-C) greatly lowers cII binding affinity, cII protein must not recognize the two T-T-G-C sequences in an identical manner.

The abortive initiation assay [McClure, W. R. (1980) Proc. Natl. Acad. Sci. USA 77, 5634-5638] was used to study the effects of mutations on the activity of the P RM promoter of phage λ in vitro . The transcription initiation properties of four mutant promoters were compared with those of wild-type P RM in the presence or absence of repressor (which activates P RM ). Two kinetic parameters were measured: k 2 , the rate constant for the transition between closed and open complexes, and K B , the equilibrium constant for the initial binding of RNA polymerase to DNA (formation of closed complexes). The primary effect of repressor on wild-type initiation was stimulation of the isomerization reaction: k 2 increased about 7-fold. Both in the presence and in the absence of repressor, prm U31 and prm E104 (changes at nucleotides -33 and -38, respectively) reduced K B significantly without affecting k 2 , indicating that these mutations affect polymerase binding but not the formation of open complexes. In contrast, prm E37 (a change at nucleotide -14) reduced k 2 significantly without affecting K B . A fourth mutation, prm E93 (at nucleotide -39), is phenotypically Prm - primarily because it causes a defect in the O B 2 operator site and, therefore, the mutant promoter is unable to respond normally to repressor. These results are consistent with the idea that the two regions of Escherichia coli promoters in which consensus sequences have been identified, the regions at nucleotides -35 and -10, may provide information for two discrete steps in transcription initiation.

The effects of cy mutations on transcription initiation at the λ P RE promoter were determined using abortive initiation analysis (McClure, 1980). In the presence of λ cll protein, which activates P RE , three mutations in the −10 region dramatically reduce k 2 , the forward rate constant for the isomerization of closed to open complexes, but only slightly affect K B , the equilibrium constant for the initial recognition by RNA polymerase to form closed complexes. In contrast, five −35 region mutations caused decreases of 30 to 150 times in K B with much smaller effects on K 2 . In the absence of cll protein, the effects of mutations in the −10 region are qualitatively similar to those observed in the presence of cll protein, although the reductions in K 2 are much less dramatic. In contrast, none of the mutants with defects in the −35 region is distinguishable from wildtype P RE in the absence of cll protein. Thus RNA polymerase may recognize different sequences in the −35 region in the absence of cll protein than in its presence.