Geoffrey Zubay

Low-usage codons in Escherichia coli, yeast, fruit fly and primates

Codon usage is compared between four classes of species, with an emphasis on characterization of low-usage codons. The classes of species analyzed include the bacterium Escherichia coli (ECO), the yeast Saccharomyces cerevisiae (YSC), the fruit fly Drosophila melanogaster (DRO), and several species of primates (PRI) (taken as a group; includes eleven species for which nucleotide sequence data have been reported to GenBank, however, greater than 90% of the sequences were from Homo sapiens). The number of protein-coding sequences analyzed were 968 for ECO, 484 for YSC, 244 for DRO, and 1518 for PRI. Three methods have been used to determine low-usage codons in these species. The first and most common way of assessing codon usage is by summing the number of time codons appear in reading frames of the genome in question. The second way is to examine the distribution of usage in different genes by scoring the number of protein reading frames in which a particular codon does not appear. The third way starts with a similar notion, but instead considers combinations of codons that are missing from the maximum number of genes. These three methods give very similar results. Each species has a unique combination of eight least-used codons, but all species contain the arginine codons, CGA and CGG. The agreement between YSC and PRI is particularly striking as they share six low-usage codons. All six carry the dinucleotide sequence, CG. The eight least-used codons in PRI include all codons that contain the CG dinucleotide sequence. Low-usage codons are clearly avoided in genes encoding abundant proteins for ECO, YSC DRO. In all species, proteins containing a high percentage of low-usage codons could be characterized as cases where an excess of the protein could be detrimental. Low codon usage is relatively insensitive to gross base composition. However, dinucleotide usage can sometimes influence codon usage. This is particularly notable in the case of CG dinucleotides in PRI.

Prebiotic synthesis of nucleotides.

If an RNA-only world preceded more complex forms oflife, then it is essential that the process wherebythe first nucleotides were made be considered. Presumably there were no enzymes and no templates tofacilitate the synthesis of the first nucleotides soanother form of chemical evolution must have beeninvolved. Answers to problems of this sort weresought vigorously in the 1960s and the early 1970s butmany issues were left unresolved. Progress made inthe last few years has added to this early work andbrings us closer to a satisfactory solution. In thisarticle key results, old and new, and some ideas as tohow further progress is likely to be made are discussed.There are reasons for optimism. Substantial progresshas been made on the synthesis of purines and ribose,phosphorylation and polyphosphorylation. Theoutstanding problems at this juncture relate to thesynthesis of ribose to the exclusion of the otheraldopentoses and to the problem of linking ribose tothe purine bases.

Studies on the Lead-Catalyzed Synthesis of Aldopentoses

The object of this work was to find an efficient means of synthesizing ribose in a manner that could be considered prebiotic. The starting point for synthesis was an aqueous solution of formaldehyde. Heretofore the most frequently used catalyst for this purpose has been calcium hydroxide. Unfortunately this system produces a wide array of products in addition to ribose which constitutes 1% or less of the final product. Attempts were made to find more mild conditions under which the formaldehyde could be reacted. Magnesium hydroxide suspensions were used for this purpose. Formaldehyde does not yield any sugars when incubated in magnesium hydroxide suspensions alone. However, if the magnesium hydroxide suspension was supplemented with doubly charged lead salts and catalytic amounts of any intermediate in the prebiotic pentose pathway, aldopentoses accounted for 30 per cent or more of the final product. The presence of lead in the incubation mixture also accelerated a number of other reactions including the interconversion of the four common aldopentoses, ribose, arabinose, lyxose and xylose.

Binding of adenine and adenine-related compounds to the clay montmorillonite and the mineral hydroxylapatite

The first living things may have consisted of no more than RNA or RNA-like molecules bound to the surfaces of mineral particles. A key aspect of this theory is that these mineral particles have binding sites for RNA and its prebiotic precursors. The object of this study is to explore the binding properties of two of the best studied minerals, montmorillonite and hydroxylapatite, for possible precursors of RNA. The list of compounds investigated includes purines, pyrimidines, nucleosides, nucleotides, nucleotide coenzymes, diaminomaleonitrile and aminoimidazole carbox-amide. Affinities for hydroxylapatite are dominated by ionic interactions between negatively charged small molecules and positively charged sites in the mineral. Binding to montmorillonite presents a more complex picture. These clay particles have a high affinity for organic ring structures which is augmented if they are positively charged. This binding probably takes place on the negatively charged faces of these sheet-like clay particles. Additional binding sites on the edges of of these sheets have a moderate affinity for negatively charged molecules.Small molecules that bind to these minerals sometimes bind independently to sites on the minerals and sometimes bind cooperatively with favorable interactions between the bound molecules.

Nucleoside Phosphorylation: A Feasible Step in the Prebiotic Pathway to RNA

Plausible prebiotic conditions for the phosphorylation of nucleosides by inorganic phosphate were reported by Lohrmann and Orgel in 1971. This reaction was carried out on heated dry films and promoted by urea. The major products formed were nucleoside-2′:3′ cyclicPs; 5′-NMPs and other derivatives were also formed. Minor modifications of the Lohrmann and Orgel system have resulted in the preferential formation of 5′-NMPs. In this modified system a 2-fold preference for phosphorylation of the 5′-OH group over the 3′(2′)-OH group was observed and the formation of other derivatives was minimized. The small amounts of bis compounds that were formed in this system could be quantitatively removed by selective binding to the mineral hydroxylapatite at moderate ionic strengths. It was also discovered that under hydrolytic conditions there was a 3:1 preference for removal of phosphates attached to the 3′-OH group over the 5′-OH group. A recycling procedure for obtaining additional 5′-NMPs from bis compounds and 3′-NMPs is proposed.

Multicopy single‐stranded DNAs with mismatched base pairs are mutagenic in Escherichia coli

Retrons are genetic elements that encode multicopy single‐stranded DNAs called msONAs. They are clonally distributed in Escherichia coli and retrons in different clones produce DNAs with different nucleotide sequences. msDNAs consist of an RNA molecule covalently linked to a single‐stranded DNA molecule. The latter contains an inverted repeat, resulting in a stem‐loop structure. In two retrons, Ec83 and Ec78, the DNA is cleaved off from the RNA. All known retrons except Ec78, have one or more mismatched base pairs in the stem‐loop structure. We found that two retrons, Ec86 and Ec83, when present in high copy numbers are mutagenic. The ratios of mutation frequencies observed in Lac indicator strains were similar to the ratios observed for a mutant defective in mismatch repair. It is known that some proteins required for mismatch repair bind to mismatched base pairs prior to carrying out repair. The similarity in the mutation frequency ratios suggested that the mutagenesis caused by msDNAs of retrons Ec86 and Ec83 might be due to seqestration of a mismatch repair protein by msDNA. Strong support for this interpretation was obtained from the finding that the msDNA produced by retron Ec78 is not mutagenic.

DNA-directed peptide synthesis I. A comparison of T2 and Escherichia coli DNA-directed peptide synthesis in two cell-free systems

Abstract Previously cell-free extracts of Escherichia coli have been used with T-bacteriophage DNA to make RNA which in turn directs a high level of peptide synthesis. Under comparable circumstances E. coli DNA or bacteriophage λ or Φ80 DNA stimulate little or no peptide synthesis. An incorporation system has been developed that works efficiently with these heretofore impotent DNAs; both RNA and peptide synthesis have been greatly increased. The main importance of this new system lies in its application for studying the activity of bacterial genes in vitro.

Evidence for the preferential binding of the catabolite gene activator protein (CAP) to DNA containing the lac promoter

Abstract The catabolite gene activator protein (CAP) is required for activation of the lac operon as well as a large number of other genes in Escherichia coli which encode catabolic enzymes. CAP-induced gene transcription occurs in the presence of cAMP; it has been shown that cAMP binds to CAP and that a tertiary complex can be formed with DNA. The cAMP-stimulated binding of CAP to DNA occurs for all DNAs thus far examined and therefore does not appear to require a specific sequence of bases on the DNA. Despite this lack of absolute specificity it has been possible to show that CAP binds with considerably greater intensity to DNA restriction fragments containing the lac operon promoter.

Synthesis of the arabinose operon regulator protein in a cell-free system.

SummaryA DNA-directed cell-free system which synthesizes L-ribulokinase coded by the L-arabinose operon, ara OIBAD, has been developed. L-arabinose is required for the expression of this operon and, in addition, cyclic AMP and guanosine-tetraphosphate are needed for optimal synthesis. The ara C gene product is also required and can be supplied either by de novo synthesis in the cell-free system or added back from extracts of whole cells. Evidence is presented that the C gene product is highly susceptible to protease attack.

Involvement of arginine in in vitro repression of transcription of arginine genes C, B and H in Escherichia coli K 12.

Abstract The combination of L-arginine and partially purified arginine repressor (the argR gene product) represses transcription of argCBH in vitro .