Mutational analysis of the guanylyltransferase component of Mammalian mRNA capping enzyme

RNA guanylyltransferase is an essential enzyme that catalyzes the second of three steps in the synthesis of the 5'-cap structure of eukaryotic mRNA. Here we conducted a mutational analysis of the guanylyltransferase domain of the mouse capping enzyme Mce1. We introduced 50 different mutations at 22 individual amino acids and assessed their effects on Mce1 function in vivo in yeast. We identified 16 amino acids as being essential for Mce1 activity (Arg299, Arg315, Asp343, Glu345, Tyr362, Asp363, Arg380, Asp438, Gly439, Lys458, Lys460, Asp468, Arg530, Asp532, Lys533, and Asn537) and clarified structure-activity relationships by testing the effects of conservative substitutions. The new mutational data for Mce1, together with prior mutational studies of Saccharomyces cerevisiae guanylyltransferase and the crystal structures of Chlorella virus and Candida albicans guanylyltransferases, provide a coherent picture of the functional groups that comprise and stabilize the active site. Our results extend and consolidate the hypothesis of a shared structural basis for catalysis by RNA capping enzymes, DNA ligases, and RNA ligases, which comprise a superfamily of covalent nucleotidyl transferases defined by a constellation of conserved motifs. Analysis of the effects of motif VI mutations on Mce1 guanylyltransferase activity in vitro highlights essential roles for Arg530, Asp532, Lys533, and Asn537 in GTP binding and nucleotidyl transfer.     

Structure-function analysis of the yeast NAD+-dependent tRNA 2'-phosphotransferase Tpt1

Tpt1 is an essential 230-amino-acid enzyme that catalyzes the final step in yeast tRNA splicing: the transfer of the 2'-PO4 from the splice junction to NAD+ to form ADP-ribose 1'-2'cyclic phosphate and nicotinamide. To understand the structural requirements for Saccharomyces cerevisiae Tpt1 activity, we performed an alanine-scanning mutational analysis of 14 amino acids that are conserved in homologous proteins from fungi, metazoa, protozoa, bacteria, and archaea. We thereby identified four residues-Arg23, His24, Arg71, and Arg138-as essential for Tpt1 function in vivo. Structure-activity relationships at these positions were clarified by introducing conservative substitutions. The activity of the Escherichia coli ortholog KptA in complementing tpt1Delta was abolished by alanine substitutions at the equivalent side chains, Arg21, His22, Arg69, and Arg125. Deletion analysis of Tpt1 shows that the C-terminal 20 amino acids, which are not conserved, are not essential for activity in vivo at 30 degrees C. These findings attest to the structural and functional conservation of Tpt1-like 2'-phosphotransferases and identify likely constituents of the active site.     

Probing the roles of conserved arginine-44 of Escherichia coli dihydrofolate reductase in its function and stability by systematic sequence perturbation analysis

Sequence perturbation analysis is a powerful method to reveal roles of an amino acid residue in function and stability of a protein. By using and improving this method, we studied roles of highly conserved Arg44 of Escherichia coli dihydrofolate reductase (DHFR) in its function and stability. Here, we introduced systematic amino acid substitutions at this position and found that all 19 kinds of amino acid substitutions were tolerated, but the mutations significantly reduced the enzymatic activity and the binding affinity toward the cofactor NADPH. Moreover, the mutational effects on the cofactor binding affinity were well correlated with those on the catalytic activity, indicating that the R44X mutations affect the catalytic activity mainly by modulating the cofactor binding affinity. On the other hand, thermal denaturation measurements showed that most mutations stabilized the protein. Comparison between the mutational effects and various amino acid indices taken from the AAindex database indicated that hydrophobicity and polarity are key determinants of amino acids favorable at this position. These results suggest that through electrostatic interactions Arg44 plays a functional role in retaining the cofactor binding affinity at the cost of the protein stability.     

Mutagenesis study on amino acids around the molybdenum centre of the periplasmic nitrate reductase from Ralstonia eutropha

Molybdenum enzymes containing the pterin cofactor are a diverse group of enzymes that catalyse in general oxygen atom transfer reactions. Aiming at studying the amino acid residues, which are important for the enzymatic specificity, we used nitrate reductase from Ralstonia eutropha (R.e.NAP) as a model system for mutational studies at the active site. We mutated amino acids at the Mo active site (Cys181 and Arg421) as well as amino acids in the funnel leading to it (Met182, Asp196, Glu197, and the double mutant Glu197-Asp196). The mutations were made on the basis of the structural comparison of nitrate reductases with formate dehydrogenases (FDH), which show very similar three-dimensional structures, but clear differences in amino acids surrounding the active site. For mutations Arg421Lys and Glu197Ala we found a reduced nitrate activity while the other mutations resulted in complete loss of activity. In spite of the partial of total loss of nitrate reductase activity, these mutants do not, however, display FDH activity.     

鸡传染性支气管炎病毒中国分离株LX4纤突蛋白基因分子特征

应用RT-PCR方法分别扩增了中国地方分离株IBV LX4 S1和S2基因并进行了基因的克隆和序列测定.结果发现,LX4 S基因由3495个核苷酸组成,编码一条1164个氨基酸残基组成的多肽.S基因编码产物裂解后形成的S1和S2亚单位分别由539和625个氨基酸残基组成.LX4 S基因推导氨基酸切割识别位点序列为HRRRR,与A2、SD/97/02和Z株相同,而与其它国内外参考毒株不同.与国内外10株已报道的具有全S基因序列的IBV参考毒株比较,LX4与国内分离毒株SD/97/02的核苷酸和氨基酸同源性最高.与32株国内外参考毒株的S1基因进化树分析比较表明,LX4与A2、SD/97/02、Z、TJ/96/02、JX/99/01和SAIBWJ等7个国内分离株在同一亚群内.在该亚群内,LX4与A2和SD/97/02亲缘关系更近,且三者在高变区和抗原表位均具有高度的同源性,而与本亚群内其它参考毒株对应的高变区和抗原表位同源性差异较大.LX4与H120 S1基因编码氨基酸的同源性虽然高于其它国外参考毒株,但同源性仍然较低,为75%.与参考毒株比较,LX4 S2基因的点突变造成其推导的氨基酸序列有11个位点发生改变,这些突变可能影响S2与S1蛋白之间的相互作用,从而影响S蛋白与特异性抗体的结合.     

Portal-drained viscera and hepatic fluxes of branched-chain amino acids do not account for differences in circulating branched-chain amino acids in rats fed arginine-deficient diets

Summary Concentrations and fluxes of amino acids across the portal-drained viscera (PDV) and liver were assessed in rats fed a meal of one of three arginine-deficient diets containing either alanine or the arginine precursors, ornithine or citrulline. A previous report included findings of seven arginine-related amino acids and indicated that only the citrulline-containing diet protected blood arginine concentrations. In the present report we extend these findings and note that the concentrations and fluxes of the non-arginine-related amino acids showed remarkable consistency across diet groups. However, total branched-chain amino acid (BCAA) concentrations of arterial blood were higher in rats fed the - Arg/+ Ala and the - Arg/+ Orn diets than in rats fed the control (+ Arg) diet. The elevated BCAA correlated with higher circulating concentrations of other essential amino acids but were inversely correlated with arginine concentrations. PDV and hepatic fluxes of BCAA were not different across diet groups, indicating that amino acid absorption and hepatic utilization of BCAA were generally comparable across diet groups. Hepatic concentrations of 14 of 22 measured amino acids, including total BCAA, were correlated with their arterial concentrations. The circulating concentrations and net PDV and hepatic fluxes of rats fed the control diet were comparable to our previous observations in fed rats and illustrate the role of the liver in utilization of diet-derived essential amino acids.Abbreviations PDV portal-drained viscera - BCAA branched-chain amino acids - SSA 5-sulfosalicylic acid - PBF portal blood flow - HBF hepatic blood flow - SELSM pooled standard errors of least squares means - TAA total amino acids - NEAA nonessential amino acids - EAA essential amino acids - LNAA large neutral amino acids Mention of a trade name, proprietary product or specific equipment does not constitute a guarantee by the US Department of Agriculture and does not imply its approval to the exclusion of other products that may be suitable.     

Identification of residues within UvrB that are important for efficient DNA binding and damage processing

The UvrB protein is the central recognition protein in bacterial nucleotide excision repair. We have shown previously that the highly conserved beta-hairpin motif in Bacillus caldotenax UvrB is essential for DNA binding, damage recognition, and UvrC-mediated incision, as deletion of the upper part of the beta-hairpin (residues 97-112) results in the inability of UvrB to be loaded onto damaged DNA, defective incision, and the lack of strand-destabilizing activity. In this work, we have further examined the role of the beta-hairpin motif of UvrB by a mutational analysis of 13 amino acids within or in the vicinity of the beta-hairpin. These amino acids are predicted to be important for the interaction of UvrB with both damaged and non-damaged DNA strands as well as the formation of salt bridges between the beta-hairpin and domain 1b of UvrB. The resulting mutants were characterized by standard functional assays such as oligonucleotide incision, electrophoretic mobility shift, strand-destabilizing, and ATPase assays. Our data indicated a direct role of Tyr96, Glu99, and Arg123 in damage-specific DNA binding. In addition, Tyr93 plays an important but less essential role in DNA binding by UvrB. Finally, the formation of salt bridges between the beta-hairpin and domain 1b, involving amino acids Lys111 bound to Glu307 and Glu99 bound to Arg367 or Arg289, are important but not essential for the function of UvrB.     

Alterations in the amino-terminal third of mouse interleukin 2: effects on biological activity and immunoreactivity

In this report, we describe plasmids that direct the expression of active mouse interleukin 2 (mIL 2) in Escherichia coli, and the use of this expression system to perform a mutational analysis of the N-terminal region of the mIL 2 protein. We found that the N-terminus was tolerant to the addition of a few amino acids, and even the addition of 20 amino acids resulted in only a modest decrease in activity of the protein. The bioactivity of mIL 2 as defined by its ability to sustain the proliferation of cloned T cells was also only minimally affected by deletion of up to 13 N-terminal amino acids, or of the entire poly-GLN stretch (amino acids 15-26). Deletion of the 30 N-terminal amino acids drastically reduced but did not abolish activity. Deletion of the 41 N-terminal amino acids completely abolished activity, whereas certain changes in the initial 37 amino acids drastically reduced the biological activity of the protein. We also analyzed the immunoreactivity of the mutant proteins with the anti-IL 2 monoclonal antibodies S4B6 and DMS-1. This analysis showed that the determinant recognized by S4B6 required that the N-terminal mIL 2 amino acids 26-45 be intact, whereas the DMS-1 determinant was located to the C-terminal side of amino acid 46.     

Cooperative Effects Between Arginine and Glutamic Acid in the Amino Acid‐Catalyzed Aldol Reaction

Catalysis of the aldol reaction between cyclohexanone and 4‐nitrobenzaldehyde by mixtures of L‐Arg and of L‐Glu in wet dimethyl sulfoxide (DMSO) takes place with higher enantioselectivity (up to a 7‐fold enhancement in the anti‐aldol for the 1:1 mixture) than that observed when either L‐Glu or L‐Arg alone are used as the catalysts. These results can be explained by the formation of a catalytically active hydrogen‐bonded complex between both amino acids, and demonstrate the possibility of positive cooperative effects in catalysis by two different α‐amino acids. Chirality 28:599–605, 2016. © 2016 Wiley Periodicals, Inc.     

Correlating amino acid conservation with function in tyrosyl-tRNA synthetase

Sequence comparisons have been combined with mutational and kinetic analyses to elucidate how the catalytic mechanism of Bacillus stearothermophilus tyrosyl-tRNA synthetase evolved. Catalysis of tRNA(Tyr) aminoacylation by tyrosyl-tRNA synthetase involves two steps: activation of the tyrosine substrate by ATP to form an enzyme-bound tyrosyl-adenylate intermediate, and transfer of tyrosine from the tyrosyl-adenylate intermediate to tRNA(Tyr). Previous investigations indicate that the class I conserved KMSKS motif is involved in only the first step of the reaction (i.e. tyrosine activation). Here, we demonstrate that the class I conserved HIGH motif also is involved only in the tyrosine activation step. In contrast, one amino acid that is conserved in a subset of the class I aminoacyl-tRNA synthetases, Thr40, and two amino acids that are present only in tyrosyl-tRNA synthetases, Lys82 and Arg86, stabilize the transition states for both steps of the tRNA aminoacylation reaction. These results imply that stabilization of the transition state for the first step of the reaction by the class I aminoacyl-tRNA synthetases preceded stabilization of the transition state for the second step of the reaction. This is consistent with the hypothesis that the ability of aminoacyl-tRNA synthetases to catalyze the activation of amino acids with ATP preceded their ability to catalyze attachment of the amino acid to the 3' end of tRNA. We propose that the primordial aminoacyl-tRNA synthetases replaced a ribozyme whose function was to promote the reaction of amino acids and other small molecules with ATP.     

Mapping the triphosphatase active site of baculovirus mRNA capping enzyme LEF4 and evidence for a two-metal mechanism

The 464-amino acid baculovirus LEF4 protein is a bifunctional mRNA capping enzyme with triphosphatase and guanylyltransferase activities. The N-terminal half of LEF4 constitutes an autonomous triphosphatase catalytic domain. The LEF4 triphosphatase belongs to a family of metal-dependent phosphohydrolases, which includes the RNA triphosphatases of fungi, protozoa, Chlorella virus and poxviruses. The family is defined by two glutamate-containing motifs (A and C), which form a metal-binding site. Most of the family members resemble the fungal and Chlorella virus enzymes, which have a complex active site located within the hydrophilic interior of a topologically closed eight stranded β barrel (the so-called ‘triphosphate tunnel’). Here we probed whether baculovirus LEF4 is a member of the tunnel subfamily, via mutational mapping of amino acids required for triphosphatase activity. We identified four new essential side chains in LEF4 via alanine scanning and illuminated structure–activity relationships by conservative substitutions. Our results, together with previous mutational data, highlight five acidic and four basic amino acids that are likely to comprise the LEF4 triphosphatase active site (Glu9, Glu11, Arg51, Arg53, Glu97, Lys126, Arg179, Glu181 and Glu183). These nine essential residues are conserved in LEF4 orthologs from all strains of baculoviruses. We discerned no pattern of clustering of the catalytic residues of the baculovirus triphosphatase that would suggest structural similarity to the tunnel proteins (exclusive of motifs A and C). However, there is similarity to the active site of vaccinia RNA triphosphatase. We infer that the baculovirus and poxvirus triphosphatases are a distinct lineage within the metal-dependent RNA triphosphatase family. Synergistic activation of the LEF4 triphosphatase by manganese and magnesium suggests a two-metal mechanism of γ phosphate hydrolysis.     

Steric and electrostatic interactions govern nanofiltration of amino acids

Crossflow nanofiltration experiments were performed to investigate the factors influencing the removal of amino acids by a commercially available polymeric thin-film composite membrane. The removals of five monoprotic (Ala, Val, Leu, Gly, and Thr), one diprotic (Asp), and one dibasic (Arg) amino acids in a range of permeate fluxes, feed pH values, and ionic strengths were analyzed using a phenomenological model of membrane transport. At any given pH and ionic strength, reflection coefficients (rejection at asymptotically infinite flux) of monoprotic amino acids increased with molar radius demonstrating the role of steric interactions on their removal. Additionally, consistent with Donnan exclusion, higher reflection coefficients were obtained when the membrane and the amino acids both carried the same nature of charge (positive or negative). In other words, both co-ion repulsion and molecular size determined amino acids removal. Importantly, the removal of effectively neutral amino acids were significantly higher than neutral sugars and alcohols of similar size demonstrating that even near their isoelectric point, zwitterionic characteristics preclude them from being considered as strictly neutral.     

Mutational analysis of human DNase I at the DNA binding interface: implications for DNA recognition,catalysis, and metal ion dependence.

Human deoxyribonuclease I (DNase I), an enzyme used to treat cystic fibrosis patients, has been systematically analyzed by site-directed mutagenesis of residues at the DNA binding interface. Crystal structures of bovine DNase I complexed with two different oligonucleotides have implicated the participation of over 20 amino acids in catalysis or DNA recognition. These residues have been classified into four groups based on the characterization of over 80 human DNase I variants. Mutations at any of the four catalytic amino acids His 134, His 252, Glu 78, and Asp 212 drastically reduced the hydrolytic activity of DNase I. Replacing the three putative divalent metal ion-coordinating residues Glu 39, Asp 168, or Asp 251 led to inactive variants. Amino acids Gln 9, Arg 41, Tyr 76, Arg 111, Asn 170, Tyr 175, and Tyr 211 were also critical for activity, presumably because of their close proximity to the active site, while more peripheral DNA interactions stemming from 13 other positions were of minimal significance. The relative importance of these 27 positions is consistent with evolutionary relationships among DNase I across different species, DNase I-like proteins, and bacterial sphingomyelinases, suggesting a fingerprint for a family of DNase I-like proteins. Furthermore, we found no evidence for a second active site that had been previously implicated in Mn2+-dependent DNA degradation. Finally, we correlated our mutational analysis of human DNase I to that of bovine DNase I with respect to their specific activity and dependence on divalent metal ions.     

Analysis of the functional modules of the tRNA 3' endonuclease (tRNase Z)

tRNA 3' processing is one of the essential steps during tRNA maturation. The tRNA 3'-processing endonuclease tRNase Z was only recently isolated, and its functional domains have not been identified so far. We performed an extensive mutational study to identify amino acids and regions involved in dimerization, tRNA binding, and catalytic activity. 29 deletion and point variants of the tRNase Z enzyme were generated. According to the results obtained, variants can be sorted into five different classes. The first class still had wild type activity in all three respects. Members of the second and third class still formed dimers and bound tRNAs but had reduced catalytic activity (class two) or no catalytic activity (class three). The fourth class still formed dimers but did not bind the tRNA and did not process precursors. Since this class still formed dimers, it seems that the amino acids mutated in these variants are important for RNA binding. The fifth class did not have any activity anymore. Several conserved amino acids could be mutated without or with little loss of activity.     

Stabilization and rational design of serine protease AprM under highly alkaline and high-temperature conditions.

Rational shift of the optimum pH toward alkalinity and enhancement of thermostability were investigated by using a thermostable extremely alkaline protease (optimum pH, 12 to 13) from the alkaliphilic and thermophilic Bacillus sp. strain B18'. The protease gene (aprM) was cloned, and the sequence analysis revealed an open reading frame of 361 amino acids that was composed of a putative signal sequence (24 amino acids), a prosequence (69 amino acids), and a mature enzyme (268 amino acids) (molecular weight, 27,664). The amino acid sequence of this protease was compared with those of other serine proteases. A direct correlation of higher optimum pH with an increase in the number of arginine residues was observed. An even more thermostable mutant enzyme was created by introducing a point mutation. When the position of the beta-turn, Thr-203, was replaced by Pro, the residual activity of this mutant enzyme at 80 degrees C for 30 min was higher than that of the wild-type enzyme (50% versus 10%). The specific activity of this mutant enzyme at 70 degrees C was 105% of that of the wild-type enzyme under nondenaturation condition. These data suggest that the higher content of Arg residues favors the alkalinity of the serine protease and that introduction of a Pro residue into the beta-turn structure stabilizes the enzyme.     

Chemical modification studies on arginine kinase: essential cysteine and arginine residues at the active site

Chemical modification was used to elucidate the essential amino acids in the catalytic activity of arginine kinase (AK) from Migratoria manilensis. Among six cysteine (Cys) residues only one Cys residue was determined to be essential in the active site by Tsou's method. Furthermore, the AK modified by DTNB can be fully reactivated by dithiothreitol (DTT) in a monophasic kinetic course. At the same time, this reactivation can be slowed down in the presence of ATP, suggesting that the essential Cys is located near the ATP binding site. The ionizing groups at the AK active site were studied and the standard dissociation enthalpy (ΔH°) was 12.38 kcal/mol, showing that the dissociation group may be the guanidino of arginine (Arg). Using the specific chemical modifier phenylglyoxal (PG) demonstrated that only one Arg, located near the ATP binding site, is essential for the activity of AK.     

Role of dietary lysine, methionine, and arginine in the regulation of hypercholesterolemia in rabbits

These experiments were conducted to see whether the hypercholesterolemia produced by a diet enriched in lysine (Lys) and methionine (Met) can be reproduced by feeding these amino acids separately, and whether dietary arginine (Arg) counteracts their hypercholesterolemic effects. Another aim was to investigate the mechanisms involved in modulations of serum cholesterol levels by these amino acids. The results of this study, which were in agreement with the results of earlier experiments in our laboratory, showed that feeding a low-fat, cholesterol-free, semipurified amino acid diet enriched with Lys + Met to rabbits caused a marked increase in serum total and low density lipoprotein cholesterol and apolipoprotein B levels, whereas a similar diet enriched in essential ketogenic amino acids (EketoAA) resulted in a more moderate increase in these parameters. Supplementing the diet with either Lys or Met alone was also less effective in inducing hypercholesterolemia than increasing levels of both amino acids. Dietary Arg partially counteracted the hypercholesterolemic effect of Lys + Met but not that of the EketoAA or of Lys and Met fed separately. The growth performance of rabbits fed the Lys + Met diet was inferior to that of those fed the other diets. Liver total phospholipid levels and the ratio of phosphatidylcholine to phosphatidylethanolamine were higher in rabbits fed the Lys + Met-enriched diet than in those animals fed a diet in which Arg was supplemented. In conclusion, our results indicate that high levels of both Lys and Met are needed to cause a maximum elevation of serum cholesterol and that the moderately antihypercholesterolemic effect of Arg is seen only when both amino acids are supplemented. They also suggest that these essential amino acids may affect cholesterol metabolism partly through alteration of liver phospholipids.     

Defensin‐like peptide3 from black solder fly: Identification,characterization, and key amino acids for anti‐Gram‐negative bacteria

An antibacterial peptide was isolated from a black soldier fly, Hermetia illucens. The molecular mass of this peptide was established as 4247.37 by matrix‐assisted laser desorption/ionization‐time of flight mass (MALD‐TOF MS) spectrometry. The amino acid sequence of the mature peptide was determined by N‐terminal sequencing using Edman degradation, combined with cDNA sequencing of the previously reported defensin‐like peptide (DLP) 3. Analysis of the minimal inhibitory concentration (MIC) revealed that DLP3 had potent activity against Gram‐positive and negative bacteria, but DLP4 had only anti‐Gram‐positive activity as previously reported. Recombinant DLP3 and DLP4 were overexpressed in Escherichia coli, and antibacterial activities were identical to DLPs purified from H. illucens hemolyph. In silico analysis revealed that only six amino acid sequences were different between DLP3 and DLP4, but antibacterial activity against Gram‐negative bacteria differed. Therefore these amino acid variants may be key amino acids (Gly‐10, Val‐18, Met‐23, Arg‐25, Asp‐32, Arg‐40) related to killing Gram‐negative bacteria.     

Percent of highly immunogenic amino acid residues forming B-cell epitopes is higher in homologous proteins encoded by GC-rich genes

We analyzed the dependence of the percent of highly immunogenic amino acid residues included in B-cell epitopes of homologous proteins on the GC-content (G+C) of genes coding for them in twenty-seven lineages of proteins (and subsequent genes), which belong to seven Varicello and five Simplex viruses. We found out that proteins encoded by genes of a high GC-content usually contain more targets for humoral immune response than their homologs encoded by GC-poor genes. This tendency is characteristic not only to the lineages of glycoproteins, which are the main targets for humoral immune response against Simplex and Varicello viruses, but also to the lineages of capsid proteins and even "housekeeping" enzymes. The percent of amino acids included in linear B-cell epitopes has been predicted for 324 proteins by BepiPred algorithm (www.cbs.dtu.dk/services/BepiPred), the percent of highly immunogenic amino acids included in discontinuous B-cell epitopes and the percent of exposed amino acid residues have been predicted by Epitopia algorithm (http://epitopia.tau.ac.il/). Immunological consequences of the directional mutational GC-pressure are mostly due to the decrease in the total usage of highly hydrophobic amino acids and due to the increase in proline and glycine levels of usage in proteins. The weaker the negative selection on amino acid substitutions caused by symmetric mutational pressure, the higher the slope of direct dependence of the percent of highly immunogenic amino acids included in B-cell epitopes on G+C.     

Role of conserved amino acids in the catalytic activity of Escherichia coli primase

The role of conserved amino acid residues in the polymerase domain of Escherichia coli primase has been studied by mutagenesis. We demonstrate that each of the conserved amino acids Arg146, Arg221, Tyr230, Gly266, and Asp311 is involved in the process of catalysis. Residues Glu265 and Asp309 are also critical because a substitution of each amino acid irreversibly destroys the catalytic activity. Two K229A and M268A mutant primase proteins synthesize only 2-nucleotide products in de novo synthesis reactions under standard conditions. Y267A mutant primase protein synthesizes both full-size and 2-nucleotide RNA, but with no intermediate-size products. From these data we discuss the significant step of the 2-nucleotide primer RNA synthesis by E. coli primase and the role of amino acids Lys229, Tyr267, and Met268 in primase complex stability.