Eukaryotes mRNA translation

Preparation of Krebs-2 translation extracts Krebs-2 extracts are an ideal system to screen for compounds that inhibit translation because they faithfully recapitulate the cap dependency and the cap-poly(A) synergism associated with eukaryotic mRNA translation (Svitkin and Sonenberg, 2004), unlike standard rabbit reticulocyte lysates (RRL) (Borman et al., 2000). Furthermore, the translation of many types of IRESes is supported in Krebs-2 extracts. The use of commercially available translation competent extracts prepared from RRL, wheat germ, and E. coli is extremely useful in assessing selectivity of inhibitors identified in primary screens. 

Different in vitro translation systems are used depending on whether the mRNA is of prokaryotic or eukaryotic origin. For eukaryotic mRNA, translation kits from reticulocyte lysates or wheat germ extract are recommended, and for prokaryotic mRNA, the E. coli 30 S supernatant, all of which are commercially available. These translation kits can be supplemented with [3H]-leudne or... 

Eukaryotic mRNA synthesis results in a pre-mRNA precursor that contains extensive amounts of excess RNA (introns) that must be precisely removed by RNA splicing to generate functional, translatable mRNA composed of exonic coding and noncoding sequences. 

Kozak M Structural features in eukaryotic mRNAs that modulate the initiation of translation. J Biol Chem 1991 266 1986. 

Figure 7.4 (a) IREs in eukaryotic mRNAs the secondary structures of ferritin and transferrin receptor IREs. (b) The IRE localization in mRNAs the translation/ribosome binding element in the 5 -UTR of ferritin mRNA is above, that of the stability/ turnover element in the 3 -UTR of transferrin receptor mRNA is below. Adapted from Theil, 1998, by courtesy of Marcel Dekker, Inc. 

Johannes, G., Carter, M. S., Eisen, M. B., Brown, P. O., and Samow, P. (1999). Identification of eukaryotic mRNAs that are translated at reduced cap binding complex eIF4F concentrations using a cDNA microarray. Proc. Natl. Acad. Sci. USA 96, 13118—13123. 

Answer E. TBP and CBP participate in eukaryotic gene transcription and mRNA translation, respectively. CAP regulates the expression of prokaryotic lactose operons. ACP is involved in fatty add synthesis. 

Most eukaryotic mRNAs have a 7-methylguanine cap at the S end, which promotes efficient translation of the message and protects it from degradation by S to S exonucleases. 

Most eukaryotic mRNAs have a 5 cap, a residue of 7-methylguanosine linked to the 5 -terminal residue of the mRNA through an unusual 5, 5 -triphosphate linkage (Fig. 26-12). The 5 cap helps protect mRNA from ribonucleases. The cap also binds to a specific capbinding complex of proteins and participates in binding of the mRNA to the ribosome to initiate translation (Chapter 27). 

Initiation in Eukaryotic Cells Translation is generally similar in eukaryotic and bacterial cells most of the significant differences are in the mechanism of initiation. Eukaryotic mRNAs are bound to the ribosome as a complex with a number of specific binding proteins. Several of these tie together the 5 and 3 ends of the message. At the 3 end, the mRNA is bound by the poly (A) binding... 

The precise manner in which proteins with zinc fingers bind to DNA differs from one protein to the next. Some zinc fingers contain the amino acid residues that are important in sequence discrimination, whereas others appear to bind DNA nonspecifically (the amino acids required for specificity are located elsewhere in the protein). Zinc fingers can also function as RNA-binding motifs—for example, in certain proteins that bind eukaryotic mRNAs and act as translational repressors. We discuss this role later (Section 28.3). 

Many Eukaryotic mRNAs Are Subject to Translational Repression... 

Removal of introns Maturation of eukaryotic mRNA usually involves the removal of RNA sequences, which do not code for protein (introns, or intervening sequences) from the primary tran script. The remaining coding sequences, the exons, are spliced together to form the mature mRNA. The molecular machine that accomplishes these tasks is known as the spliceosome. [Note A few eukaryotic primary transcripts contain no introns. Others con tain a few introns, whereas some, such as the primary transcripts for the a-chains of collagen, contain more than fifty intervening sequences that must be removed before mature mRNA is ready for translation.]... 

The pathway of protein synthesis translates the three-letter alphabet of nucleotide sequences on mRNA into the twenty-letter alphabet of amino acids that constitute proteins. The mRNA is translated from its 5 -end to its 3 -end, producing a protein synthesized from its amino-terminal end to its carboxyl-terminal end. Prokaryotic mRNAs often have several coding regions, that is, they are polycistronic (see p. 420). Each coding region has its own initiation codon and produces a separate species of polypeptide. In contrast, each eukaryotic mRNA codes for only one polypeptide chain, that is, it is monocistronic. The process of translation is divided into three separate steps initiation, elongation, and termination. The polypeptide chains produced may be modified by posttranslational modification. Eukaryotic protein synthesis resembles that of prokaryotes in most details. [Note Individual differences are mentioned in the text.]... 

Initiation The components of the translation system are assembled, and mRNA associates with the small ribosomal subunit. The process requires initiation factors. In prokaryotes,a purine-rich region (the Shine-Dalgarno sequence) of the mRNA base-pairs with a complementary sequence on 16S rRNA, resulting in the positioning of the mRNA so that translation can begin. The 5 -cap on eukaryotic mRNA is used to position that structure on the ribosome. The initiation codon is 5 -AUG-3. ... 

A poly(A) "tail" consisting of -250 residues of adenylic acid is added next by poly(A) polymerase, a component of an enzyme complex that also cleaves the RNA chains.545 57111 Most eukaryotic mRNA is polyadenylated with the exception of that encoding histones. The function of the poly(A) is unclear. It is needed for transport of mRNA out of the nucleus, but it does confer a greatly increased stability to the mRNA in the cytoplasm where the adenylate irnits are gradually removed.307 308 In contrast, in chloroplasts and plant mitochondria polyadenylation is required for rapid degradation of mRNA.571c d Polyadenylation may also increase the efficiency of translation.572 Polyadenylation occurs rapidly within -1 min after transcription is completed. 

The 5 and 3 UTRs of eukaryotic mRNA are crucial for the regulation of gene expression by controlling mRNA translational efficiency, stability, and localization. The 5 cap and poly A (pA), found on almost all eukaryotic mRNAs, stimulate translation initiation and stabilize mRNA in synergy. However, for in vitro translation, the use of 5 cap and pA can be a major problem, as described Chapter 11 by Endo and Sawasaki. These problems have been effectively overcome by optimizing the 5 and 3 UTRs (12). 

Pelletier, J. and Sonnenberg, N. (1988) Internal initiation of translation of eukaryotic mRNA directed by a sequence derived from poliovirus RNA. Nature 334, 320-325. 

Eukaryotic mRNA processing is much more complex and has many consequences for gene expression. Most obviously, many eukaryotic genes contain introns, which are found in the primary transcript. For the mRNA to be translated into a useful protein, some way to remove them from the transcript and still preserve the coding... 

The 5 untranslated region (5 UTR) is the region of the mRNA transcript that is located between the cap site and initiation codon. The linkage between methylated G residue and a 5 to 5 triphosphate bridge is known as the cap structure, which is essential for efficient initiation of protein synthesis. The 5 UTR is known to influence mRNA translation efficiency. In eukaryotic cells, initiation factors first interact with the 5 cap structure and prepare the mRNA by unwinding its secondary structure. An efficient 5 UTR is usually moderate in length, devoid of strong secondary structure, devoid of upstream initiation codons, and has AUG within an optimal context. 

Colman A (1984) Translation of eukaryotic mRNA in Xenopus oocytes. In Hames D, Higgins S (eds) Transcription and Translation - A Practical Approach. IRL Press, Oxford, p 271-301... 

What major structural requirement for initiation of translation of eukaryotic mRNA docs not exist for bacterial mRNA ... 

During the processing of primary transcripts destined for mRNA formation in eukaryotes, a methylated guanine cap is attached to the 5 end. This is essential for efficient initiation of translation of the mature eukaryotic mRNA and does not occur in prokaryotes. 

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