Transcription eukaryotic protein synthesis

The binding of interferons to their receptors induces a rapid increase in the transcription of particular genes and synthesis of corresponding proteins.196 202 One of the proteins induced is a double-stranded RNA-activated 2 -5 A synthase, which polymerizes ATP to a series of 2 -5 linked oligonucleotides containing triphosphates at the 5 termini.202-204 Double-stranded RNA is uncommon except in replicating viruses, and it is thought that the activation by dsRNA is related to establishment of an antiviral state. Another interferon-induced enzyme is the small subunit of eukaryotic protein synthesis initiation factor eIF-2. 

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. 

In this section, we describe the three basic stages of protein synthesis initiation, elongation, and termination. These three processes are fairly similar between prokaryotes and eukaryotes, with the two exceptions being that more protein factors have been identified as necessary for eukaryotic protein synthesis, and that transcription and translation are physically linked in prokaryotes but not in eukaryotes. Note that the reactions will be schematized as a single ribosome transversing the mRNA, but as shown in Figure 26.3, translation actually occurs on polyribosomes. 

Comparison of DNA replication, DNA transcription and protein synthesis in eukaryotes and prokaryotes... 

The mechanism of protein synthesis in eukaryotic cells is also essentially the same as in bacteria. However, since eukaryotic protein synthesis occurs in the cytoplasm, the processes of transcription and translation are not as closely coupled as they are in bacteria. In eukaryotes the initiating methionine is not formylated, but is attached to a different form of tRNA from that involved in incorporating internal methionine residues. 

The protein synthesis machinery reads the RNA template starting from the 5 end (the end made first) and makes proteins beginning with the amino terminus. These directionalities are set up so that in prokaryotes, protein synthesis can begin even before the RNA synthesis is complete. Simultaneous transcription-translation can t happen in eukaryotic cells because the nuclear membrane separates the ribosome from the nucleus. 

Transcription is the first step in the complicated and energy-intensive pathway of protein synthesis, so much of the regulation of protein levels in both bacterial and eukaryotic cells is directed at transcription, particularly its early stages. In Chapter 28 we describe many mechanisms by which this regulation is accomplished. 

Nearly all of the RNA of the cell is synthesized (transcribed) in the nucleus, according to the instructions encoded in the DNA. Some of the RNA then moves out of the nucleus into the cytoplasm where it functions in protein synthesis and in some other ways. Many eukaryotic genes consist of several sequences that may be separated in the DNA of a chromosome by intervening sequences of hundreds or thousands of base pairs. The long RNA transcripts made from these split genes must be cut and spliced in the nucleus to form the correct messenger RNA molecules which are then sent out to the ribosomes in the cytoplasm. 

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. 

The absence of a nuclear membrane is a characteristic of bacteria that has a profound effect on transcription. Bacterial transcripts are processed rapidly, and their 5 ends often enter ribosomes and are directing protein synthesis, while the 3 ends of the genes are still being transcribed. In contrast, most eukaryotic RNA transcripts must be processed and transported out of the nucleus before they can function. As consequence, many aspects of the control of transcription differ between prokaryotes and eukaryotes. 

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... 

Another way in which gene expression is regulated is by translational control, where the rate of protein synthesis is controlled at the point of transcription of mRNA into polypeptides (Appendix 5.6). Generally, the majority of the control mechanisms in bacteria is at the transcriptional level. Translational control is less well understood and appears to be a secondary mechanism in bacteria, but it is thought to be very important in eukaryotic organisms. 

The cap protects the 5 end of the primary transcript against attack by ribonu-cleases that have specificity for 3 5 phosphodiester bonds and so cannot hydrolyze the 5 5 bond in the cap structure. In addition, the cap plays a role in the initiation step of protein synthesis in eukaryotes. Only RNA transcripts from eukaryotic protein-coding genes become capped prokaryotic mRNA and eukaryotic rRNA and tRNAs are uncapped. 

Chloramphenicol acetyl transferase (CAT). This bacterial enzyme was the first reporter protein used for studying the transcriptional activity of eukaryotic regulatory sequences (Gorman et al., 1982). CAT inactivates chloramphenicol, an inhibitor of prokaryotic protein synthesis, by converting it to the mono- or di-acetylated species. Measurement of CAT activity requires a 14C-radiolabeled chloramphenicol or acetyl-CoA and, therefore, an additional step is neccessary to separate the radio-labeled reactant from the product. Novel detection methods based on fluorescent substrates or ELISA assays, which do not use radiolabeled reagents, have been described more recently (Bullock and Gorman, 2000). 

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. 

Both pro- and eukaryotic cells have three types of RNA. The largest is messenger RNA (mRNA), whose molecular weight can be as high as 106. It carries genetic information from DNA to the protein synthesis machinery. It is a product of DNA transcription, and it is translated (expressed) into a protein via... 

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. 

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