Coupled transcription and translation

Operons that produce the enzymes of amino acid synthesis have a regulatory circuit called attenuation, which uses a transcription termination site (the attenuator) in the mRNA. Formation of the attenuator is modulated by a mechanism that couples transcription and translation while responding to small changes in amino acid concentration. 

In addition to mRNA and tRNA, the third major class of RNA molecule required for protein synthesis is rRNA. Together with as many as 70 ribosomal proteins, rRNA folds into a two-subunit macromolecule complex called a ribosome (Chapter 5). In bacteria, the ribosomes attach to mRNA as it is being synthesized, thereby coupling transcription and translation. In eukaryotes, protein synthesis occurs in the cytoplasm, either by free ribosomes in the cytosol or by membrane-bound ribosomes associated with the endoplasmic reticulum. The differences between prokaryotic and eukaryotic protein synthesis are illustrated in Figure 26.3. 

Iborra F.J., Jackson D.A., Cook P.R. (2001). Coupled transcription and translation within nuclei of mammalian cells. Science 293 1139-1142. 

It has recently been found that there is some coupled transcription and translation in the nucleus of eukaryotic cells. 

Iborra, F. J., D. A. Jackson, and P. R. Cook. Coupled Transcription and Translation within Nuclei of Mammalian Cells. Science293, 1139-1142 (2001). [The primary research showing translation in the nucleus.]... 

The authors next turn to the more complex process of transcription in eukaryotes. The impossibility of coupling transcription and translation in eukaryotes as it occurs in prokaryotes (because of eukaryotic subcellular separation in the nucleus and cytoplasm) is pointed out. The three eukaryotic RNA polymerases that carry out transcription are described and related to the kinds of RNA they synthesize. The role of the eukaryotic TATA box and the TATA-box-binding protein in basal transcription are explained, as are other eukaryotic promoters and enhancers and some of the proteins... 

Bossart, W., Nuss, D. L., and Paoletti, E., 1978, Effect of UV-irradiation on the expression of vaccinia virus gene products synthesized in a cell-free system coupling transcription and translation, J. Virol. 26 673. 

Pelham, H. R. B., 1977, Use of coupled transcription and translation to study mRNA production by vaccinia cores. Nature 269 532. 

This tRNA (BODIPY-lysyl-tRNA > ) was recently used to demonstrate the presence of coupled transcription and translation within the nuclei of mammalian cells. This was achieved by incubating permeabfiized mammalian cells with lysyl-transfer RNA tagged with BODIPY (BODIPY-lysyl-tRNA > ). Although most nascent polypeptides were found in the cytoplasm, some were found in discrete nuclear sites known as transcription factories. This coupling is simply explained, if nuclear ribosomes translate nascent transcripts as those transcripts emerge from still-engaged RNA polymerases, much as they do in bacteria. 

In bacteria, transcription and translation are tightly coupled. Messenger RNAs are synthesized and translated in the same 5 — 3 direction. Ribosomes begin translating the 5 end of the mRNA before transcription is complete (Fig. 27-28). The situation is quite different in eukaryotic cells, where newly transcribed mRNAs must leave the nucleus before they can be translated. 

FIGURE 27-28 Coupling of transcription and translation in bacteria. The mRNA is translated by ribosomes while it is still being transcribed from DNA by RNA polymerase. This is possible because the mRNA in bacteria does not have to be transported from a nucleus to the cytoplasm before encountering ribosomes. In this schematic diagram the ribosomes are depicted as smaller than the RNA polymerase. In reality the ribosomes (Mr 2.7 X 105) are an order of magnitude larger than the RNA polymerase (Mr 3.9 X 105). 

Regulation at the level of translation assumes a much more prominent role in eukaryotes than in bacteria and is observed in a range of cellular situations. In contrast to the tight coupling of transcription and translation in bacteria, the transcripts generated in a eukaryotic nucleus... 

Figure 7.15. Functions of cold-shock proteins (Csp s) as RNA chaperones. The model shows how Csp s assist in coupling transcription to translation. Cold-shock proteins bind relatively weakly to nascent mRNA extending from the RNA polymerase complex (RNAP) and maintain the mRNA in a linear form that can be bound to ribosomes and translated into protein. Under nonstressful conditions, the weakly binding Csp s are present at adequate concentrations to perform this chaperoning function. However, during cold stress, the propensity for RNA to form secondary structures that block translation becomes greater. This necessitates that a higher level of Csp s be present in the cell, to ensure that chaperoning of mRNA is effective. (Figure modified after Graumann and Marahiel, 1998.)...

Prokaryotic ribosomes attach to the nascent mRNA while it is still being transcribed. Because transcription and translation are coupled, prokaryotic mRNAs undergo little modification and processing before being used as templates for protein synthesis. Prokaryotic tRNA and rRNA are transcribed in units larger than those ultimately used and must be processed to generate the functional molecules. The processing of these and the eukaryotic primary transcripts, almost all of which require modification, is discussed in a later section. 

Attenuation of transcription is relatively common in bacterial gene expression and occurs in at least six other amino acid biosynthetic pathways (histidine, threonine, phenylalanine, leucine, isoleucine, and valine). These are also based on the coupling of transcription and translation. 

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. 

We turn now to transcription in eukaryotes, a much more complex process than in prokaryotes. In eukaryotes, transcription and translation take place in different cellular compartments transcription takes place in the membrane-bounded nucleus, whereas translation takes place outside the nucleus in the cytoplasm. In prokaryotes, the two processes are closely coupled (Figure 28.15). Indeed, the translation of bacterial mRNA begins while the transcript is still being... 

Figure 28.15. Transcription and Translation. These two processes are closely coupled in prokaryotes, whereas they are spacially and temporally separate in eukaryotes. (A) In prokaryotes, the primary transcript serves as mRNA and is used immediately as the template for protein synthesis. (B) In eukaryotes, mRNA precursors are processed and spliced in the nucleus before being transported to the cytosol for translation into protein. [After J. Darnell, H. Lodish, and D. Baltimore. Molecular Cell Biology, 2d ed. (Scientific American Books, 1990), p. 230.]...

In prokaryotes, transcription and translation are closely coupled. Several ribosomes can simultaneously translate an mRNA, forming a polysome. 

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