Transcription and Translation in Eukaryotes

Until recendy, the dogma of eukaryotic translation was that it was physically separated from transcription. Transcription occurred in the nucleus, and mRNA was then exported to the cytosol for translation. Although this system is accepted as the normal process, recent evidence has shown that the nucleus has all of the components (mRNA, ribosomes, protein factors) necessary for translation. In addition, evidence shows that, in isolated test systems, proteins are translated in the nucleus. The authors of the most recent work suggest that 10%-15% of the cell s protein synthesis occurs in the nucleus. See the articles by Hentze and by Iborra et al. cited in the bibliography at the end of this chapter. 

Eukaryotic translation involves many more protein factors than the corresponding translation in prokaryotes. 

Both the 5 cap and the 3 poly A tail are involved in orienting the ribosome close to the correct AUG used as the start codon. There is no Shine-Dalgarno sequence in eukaryotic mRNA. 

Once bound, the ribosome moves down the mRNA scanning for the correct AUG until it finds one that is in the correct context, which is identified by a small mRNA sequence around the AUG called a Kozak 

Eukaryotic chain elongation is similar to the prokaryotic counterpart. With chain termination, there is only one release factor that binds to all three stop codons. 

---

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. 

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... 

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 4.1 Differences in transcription and translation in bacteria and eukaryotes (Watson, 1992).

This flow of information depends on the genetic code, which defines the relation between the sequence of bases in DNA (or its mRNA transcript) and the sequence of amino acids in a protein. The code is nearly the same in all organisms a sequence of three bases, called a codon, specifies an amino acid. There is another step, between transcription and translation, in the expression of most eukaryotic genes, which are mosaics of nucleic acid sequences called introns and exons. Both are transcribed, but introns are cut out of newly synthesized RNA molecules, leaving mature RNA molecules with continuous exons. The existence of introns and exons has crucial Implications for the evolution of proteins. 

A FIGURE 4-12 Comparison of gene organization, transcription, and translation in prokaryotes and eukaryotes. 

A number of differences between eukaryotes and prokaryotes affect the processes of replication, transcription, and translation, in addition to the content of their DNA. Eukaryotic DNA is complexed with histones, and prokaryotic DNA is not. In eukaryotic cells, the process of transcription, which occurs in the nucleus, is separated by the nuclear envelope from the process of translation (protein synthesis from the mRNA template), which occurs in the cytoplasm. Because prokaryotes lack nuclei, the processes of transcription and translation occur simultaneously. Transcription of bacterial DNA requires only one promoter per operon. In contrast, human DNA requires one promoter for each gene. 

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

The existence of an extrachromosomal genome localized in mitochondria as well as machinery for transcription and translation in these cell organelles is now well established. There has been considerable speculation on the origin of mitochondria and the idea of a symbiosis between eukaryotic cells and prokaryotic organisms is favored by many scientists (e.g., Nass, 1971, Fridovich, 1974, but cf. Raff and Mahler, 1972). 

FIGURE 11.24 The properties of mRNA molecules in prokaryotic versus eukaryotic cells during transcription and translation. 

Colman A. 1984. Translation of eukaryotic messenger RNA in Xenopus oocytes. Transcription and translation a practical approach. Rickwood D, Hames BD, editors. Washington, DC Oxford University Press pp. 271-302. 

Attenuation is not used as a regulatory mechanism in eukaryotes, because transcription and translation are independent events and occur in different subcellular locations. 

In eukaryotic cells transcription and translation occur in two distinct temporal and spacial events, whereas in prokaryotic cells they occur in one step. As humans have eukaryotic cells, we will look at this process. Transcription occurs on DNA in the nucleus and translation occurs on ribosomes in the cytoplasm. 

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. 

We begin by examining the interactions between proteins and DNA that are the key to transcriptional regulation. We next discuss the specific proteins that influence the expression of specific genes, first in prokaryotic and then in eukaryotic cells. Information about posttranscriptional and translational regulation is included in the discussion, where relevant, to provide a more complete overview of the rich complexity of regulatory mechanisms. 

A major goal in recombinant DNA technology is the production of useful foreign proteins by bacteria, yeast, or other cultured cells. Protein synthesis depends upon both transcription and translation of the cloned genes and may also involve secretion of proteins from the host cells. The first step, transcription, is controlled to a major extent by the structures of promoters and other control elements in the DNA (Chapter 28). Since eukaryotic promoters often function poorly in bacteria, it is customary to put the cloned gene under the control of a strong bacterial or viral X promoter. The latter include the X promoter PL (Fig. 28-8) and the lac (Fig. 28-2) and trp promoters of E. coli. These are all available in cloning vehicles. 

In bacteria transcription and translation are closely linked. Polyribosomes may assemble on single DNA strands as shown in Fig. 28-5. It has often been assumed that RNA synthesis occurs on loops of DNA that extend out into the cytosol. However, recent studies indicate that most transcription occurs in the dense nucleoid and that assembly of ribosomes takes place in the cytosol.2683 In a similar way eukaryotic transcription occurs in the nucleus and protein synthesis in the cytosol. Nevertheless, some active ribosomes are present in the nucleus.26813... 

We have stated that translation begins before transcription is completed in prokaryotes. The situation is quite different in eukaryotes, where transcription and translation occur in different cellular compartments separated by the nuclear membrane. Large precursors of mRNA are synthesized in the nucleus these become complexed with proteins to form ribonucleoprotein particles which are modified and processed to form smaller mRNAs that become transported across the nuclear membrane to the cytoplasm. 

SELEX has also allowed the characterization of the RNA hairpin, which constitutes the iron responsive element (IRE) recognized by the iron regulatory factor (IRF) protein to post-transcriptionally regulate translatability and decay of mRNAs involved in iron import and storage in eukaryotic cells (Henderson et al., 1994). 

---