Prokaryotes translation initiation

Brandi, L., Fabbretti, A., La Teana, A., Abbondi, M., Losi, D., Donadio, S., and Gualerzi, C. O. (2006b). Specific, efficient, and selective inhibition of prokaryotic translation initiation by a novel peptide antibiotic. Proc. Nat. Acad. Sci. USA 103, 39-44. 

Abbreviations aa-tRNA Amino-acyl tRNA eLF Eukaryotic translation initiation factor IF Prokaryotic translation initiation factor eEF Eukaryotic translation elongation factor EF Prokaryotic translation elongation factor eRF Eukaryotic translation termination factor (release factor) RF Prokaryotic translation release factor RRF Ribosome recycling factor Rps Protein of the prokaryotic small ribosomal subunit Rpl Protein of the eukaryotic large ribosomal subunit S Protein of the prokaryotic small ribosomal subunit L Protein of the prokaryotic large ribosomal subunit PTC Peptidyl transferase center RNC Ribosome-nascent chain-mRNA complex ram Ribosomal ambiguity mutation RAC Ribosome-associated complex NMD Nonsense-mediated mRNA decay... 

The Shine-Salgano interaction is a base pairing interaction that occurs during translation initiation in prokaryotes between the Shine-Dalgarno sequence on messenger RNA (mRNA) and the anti-Shine-Dalgamo... 

This chapter presents methods and protocols suitable for the identification and characterization of inhibitors of the prokaryotic and/or eukaryotic translational apparatus as a whole or targeting specific, underexploited targets of the bacterial protein synthetic machinery such as translation initiation and amino-acylation. Some of the methods described have been used successfully for the high-throughput screening of libraries of natural or synthetic compounds and make use of model universal mRNAs that can be translated with similar efficiency by cellfree extracts of bacterial, yeast, and HeLa cells. Other methods presented here are suitable for secondary screening tests aimed at identifying a ... 

Gribskov, M. (1992). Translational initiation factors IF-1 and eIF-2 alpha share an RNA-binding motif with prokaryotic ribosomal protein SI and polynucleotide phosphoryl-ase. Gene 119, 107-111. 

The first phase of translation, initiation, involves several steps. First, two proteins, initiation factors IF-1 and IF-3, bind to the 30 S subunit (1). Another factor, IF-2, binds as a complex with GTP (2). This allows the subunit to associate with the mRNA and makes it possible for a special tRNA to bind to the start codon (3). In prokaryotes, this starter tRNA carries the substituted amino acid N-formylmethionine (fMet). In eukaryotes, it carries an unsubstituted methionine. Finally, the 50 S subunit binds to the above complex (4). During steps 3 and 4, the initiation factors are released again, and the GTP bound to IF-2 is hydrolyzed to GDP and Pj. 

Initiation of protein synthesis involves the assembly of the components of the translation system before peptide bond formation occurs. These components include the two ribosomal subunits, the mRNA to be translated, the aminoacyl-tRNA specified by the first codon in the message, GTP (which provides energy for the process), and initiation factors that facilitate the assembly of this initiation complex (see Figure 31.13). [Note In prokaryotes, three initiation factors are known (IF-1, IF-2, and IF-3), whereas in eukary- otes, there are at least ten (designated elF to indicate eukaryotic origin).] There are two mechanisms by which the ribosome recognizes the nucleotide sequence that initiates translation ... 

As we have indicated, the codon AUG is the only one generally used to specify methionine, but it serves a dual function in that it is also used to initiate translation. Occasionally, GUG and UUG are also read as an initiating codon in bacteria, but in internal positions these codons are always read as valine and leucine, respectively. In eukaryotes, initiation at codons other than AUG is much less frequent than in prokaryotes. Weak initiation occasionally occurs at GUG, CUG, and ACG codons in eukaryotic systems. The UGA triplet also serves a dual function it is usually recognized as a stop, but on occasion it serves as a codon for selenocys-teine (box 29A). 

Compare the translation initiation signals in prokaryotic and eukaryotic systems, and describe those features of each type of mRNA that determine the frequency with which a particular message is translated. What consequences do these differences have for gene organization in the two systems ... 

Pestova, T. V., Shatsky, I. N., Fletcher, S. P., Jackson, R. J., and Hellen, C. U. (1998). A prokaryotic-like mode of cytoplasmic eukaryotic ribosome binding to the initiation codon during internal translation initiation of hepatitis C and classical swine fever virus RNAs. Genes Dev. 12, 67—83. 

Translation Initiation Site. In eukaryotes, if the transcription start site is known, and there is no intron interrupting the 5 UTR, Kozak s rule (Kozak, 1996) probably will locate the correct initiation codon in most cases. Splicing is normally absent in prokaryotes, yet because of the existence of multicitronic operons, promoter location is not the key information. Rather, the key is reliable localization of the ribosome binding site. The TATA sequence about 30 bp from the transcription start site may be used as a possible resource. 

Figure 23-3 Schematic diagram of translational initiation in prokaryotes.

Another difference between prokaryotic and eukaryotic translation is the nucleic acid sequences that recruit the small ribosomal subunit to the mRNA. As stated earlier, the Shine-Delgarno sequence interacts with the 16S rRNA of the 3 OS ribosomal subunit in the prokaryotic system, the critical step in translation initiation. In the eukaryotic... 

Eukaryotic translation initiation is far more complicated than the prokaryotic system described earlier. To begin the process, two initiation factor... 

The initiation codon, usually an AUG, signals the start of translation, and a termination codon marks the end of the translated region. In the analysis of prokaryotic DNA sequences, the signals include the transcriptional and translational initiation sites, the ribosome-binding site, and the transcriptional and translational termination sites. Due to the interrupted nature of the eukaryotic genes, the signals include the translation initiation sites, the intron/exon boundaries (splice sites), translational termination sites, and the polyadenylation sites. 

The mRNA required for in vitro translation can itself be produced by in vitro synthesis. Commercially available kits allow DNA cloned downstream of T7-, T3 or SP6-promoters to be transcribed effectively in vitro by the relevant RNA polymerases. In coupled transcription-translation, it should be remembered that translation of eukaryotic mRNA requires a 5 cap upstream of the initiation codon, and similarly, for prokaryotic translation there should be an appropriately positioned ribosome binding site. Commercial kits are also available for combined in vitro transcription and translation. 

Figure 29.27. Translation Initiation in Prokaryotes. Initiation factors aid the assembly first of the 308 initiation complex and then of the 70S initiation complex.

Eukaryotic Protein Synthesis Differs from Prokaryotic Protein Synthesis Primarily in Translation Initiation... 

In prokaryotic translation, where die initiation codon interacts with the anticodon of f-Met-tRNAfmet, what is the sequence of the anticodon ... 

Eukaryotes utilize many more initiation factors than do prokaryotes, and their interplay is much more intricate. The prefix elF denotes a eukaryotic initiation factor. For example, eIF-4E is a protein that binds directly to the 7-inethylguanosine cap (p. 846), whereas eIF-2, in association with GTP, delivers the met-tRNA to the ribosome. The difference in initiation mechanism between prokaryotes and eukaryotes is, in part, a conseciuence of the ence in RNA processing. The 5 end of mRNA is readily available to ribosomes immediately after transcription in prokaryotes. In contrast, pre-mRNA must be processed and transported to the cytoplasm in eukaryotes before translation is initialed. The 5 cap provides an easily recognizable starting point. In addition, the complexity of eukaryotic translation initiation provides another mechan ism for regulation of gene expression that we shall explore further in Chapter 31. 

Just before the AUG start codon, prokaryotic mRNAs have a common sequence called the Shine-Dalgamo sequence. Some variations on the sequence are shown in Table 27,3. The Shine-Dalgamo sequences are complementary to a sequence contained in the 16S ribosomal RNA (rRNA) (Table 27.3) and help align the mRNA with the ribosome to properly orient the molecules for translation initiation. 

See also The Genetic Code, Structure of tRNAs, Initiation of Translation, Prokaryotic Translation Regulation, Lactose Operon Regulation (from Chapter 26), Promoter Organization... 

---