Escherichia coli initiation factors

Although AUG is the initiation codon normally used in bacteria, studies of binding of fMet-tRNAf to ribosomes (Clarck and Marcher, 1966) or translation of synthetic polynucleotides (Thach et ai, 1966) have revealed that GUG and, to a lesser extent, UUG are also functional initiation codons. A single case of initiation at an AUU codon is also known for the Escherichia coli initiation factor IF-3 protein (Sacerdot et ai, 1982). By contrast, a compilation of over 200 ribosome binding site sequences in eukaryotic mRNA (Kozak, 1981a, 1983) has yielded only AUG as initiation codon. It appears, therefore, that initiation of eukaryotic translation occurs exclusively at AUG codons. 

Edery, I., Altmann, M., and Sonenberg, N. (1988). High-level synthesis in Escherichia coli of functional cap-binding eukaryotic initiation factor eIF-4E and affinity purification using a simplified cap-analog resin. Gene 74, 517-525. 

Escherichia coli genes are transcribed once per second, others less than once per cell generation. Much of this variation is due to differences in promoter sequence. In the absence of regulatory proteins, differences in promoter sequences may affect the frequency of transcription initiation by a factor of 1,000 or more. Most E. coli promoters have a sequence close to a consensus (Fig. 28-2). Mutations that result in a shift away from the consensus sequence usually decrease promoter function conversely, mutations toward consensus usually enhance promoter function. 

Alternative Sigma Factors Trigger Initiation of Transcription at Different Promoters Elongation of the Transcript Termination of Transcription Comparison of Escherichia coli RNA Polymerase with DNA Poll and PolIII... 

Although the mechanism whereby proteins are synthesized is the same in all living forms, classical distinctions exist between eucaryal (post-transcriptional) and bacterial (co-transcriptional) translation. In-frame read-out of (usually polycistronic) bacterial mRNAs is established via Shine-Dalgarno mRNA 16S-rRNA recognition mechanisms polypeptide synthesis is initiated by a formylated methionine and the initiation reactions are assisted by a limited number of protein factors (three in Escherichia coli) that primarily influence kinetic parameters [3],... 

Several fluorescence and biochemical experiments reveal the detailed mechanism of translation and the contribution of KPR towards the fidelity of protein synthesis. The incorporation of an amino acid into the peptide is composed of two consecutive processes initial selection of tRNA at the A site of the ribosome followed by KPR [23]. Various factors affect the initial selection of tRNA such as the HB energy between the codon-anticodon base pairs, the specific interactions between the large subunit of the ribosome and aa-tRNA, etc. The contribution of the initial selection step to the overall error fraction for Escherichia coli is observed to be -1/6, compared to the overall error fraction -7 x 10 for cognate and near-cognate anticodons. As a consequence the contribution of KPR is expected to be 1/24 i.e. -80% of the observed fidelity comes from the KPR. The translation process occurs through the following mechanism (see Figure 13.2). 

We have mentioned how DNA polymerase I from Escherichia coli extends the DNA chain, but how does it initiate the chain A clue to the mechanism was obtained when it was observed that rifampicin, an inhibitor of RNA polymerase, also inhibits the conversion of a phage single stranded into a double stranded DNA circle. This suggested that DNA synthesis is initiated by RNA priming. It is now established that the initiation of DNA synthesis by DNA polymerase I requires RNA and DNA polymerase, ribose and deoxyribose triphosphates, an unwinding protein and other factors whose roles are still unclear. The initiation step seems to involve a transcriptional operation by RNA polymerase. The newly synthesized RNA serves as primer for DNA polymerase I, yielding an RNA-DNA complex and the primary RNA is excised by a nuclease [236, 237]. 

The mechanism whereby RNA is translated into protein is complex, and the cell devotes considerable resources to the translational machinery. The components include 20 different amino acids, transfer RNAs, aminoacyl-tRNA synthetases, ribosomes, and a number of protein factors which cycle on and off the ribosomes and facilitate various steps in initiation of translation, elongation of the nascent polypeptide chain, and termination of synthesis with release of the completed polypeptide from the ribosome. The process depends on a supply of energy provided by ATP and GTP. The rate of protein synthesis is typically in the range of 6 (immature red blood cells of the rabbit) to 20 Escherichia coli growing optimally) peptide bonds per sec. at 37°C. 

Noll, M., Noll, H., Lingrel, J. B. Initiation factor IF-3-dependent binding of Escherichia coli ribosomes and N-formylmethionine transfer-RNA to rabbit globin messenger. Proc. nat. Acad. Sci. (Wash.) 69, 1843-1847 (1972). 

The major polyamines in Escherichia coli cells are putrescine, spermidine, and cadaverine this bacterium does not synthesize spermine. In eukaryotes, the lysine residue of the precursor of eukaryotic translation initiation factor 5 A (elF5A) is hypusinated by the sequmtial reactions of two enzymes using spermidine. 

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