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The mRNA contains several cistrons, each of which codes for a protein.

In higher organisms operons are absent and neighboring genes are not co-transcribed. Each individual gene is transcribed separately to give an individual molecule of RNA. Apart from a few exceptional cases, each molecule of eukaryotic mRNA only carries a single protein coding sequence. Furthermore, eukaryotic mRNA does not make use of the Shine-Dalgarno sequence. Instead, the front (5'-end) of the messenger RNA molecule is recognized by its cap structure. Consequently, in eukaryotes only the first open reading frame would normally be translated, even if multiple open reading frames were present (see below for further discussion).

Transcription and Translation Are Coupled in Bacteria

When mRNA is transcribed from the original DNA template, its synthesis starts at the 5'-end. The mRNA is also read by the ribosome starting at the 5'-end. In prokaryotic cells, the chromosome and ribosomes are all in the same single cellular compartment.

Some Ribosomes Become Stalled and Are Rescued 217

^ Transcription initation site

RNA polymerase

Growing polypeptide

^ Transcription initation site

RNA polymerase

Growing polypeptide

Translation

FIGURE 8.21 Coupled Transcription-Translation in Bacteria

Even as the DNA is being transcribed to give mRNA, ribosomes sequentially attach to the growing mRNA and initiate protein synthesis.

Translation

FIGURE 8.21 Coupled Transcription-Translation in Bacteria

Even as the DNA is being transcribed to give mRNA, ribosomes sequentially attach to the growing mRNA and initiate protein synthesis.

In prokaryotes, the ribosomes can begin to translate a message before the RNA polymerase has finished transcribing it.

Therefore the ribosomes can start translating the message before the synthesis of the mRNA molecule has actually been finished. The result is that partly finished mRNA, still attached to the bacterial chromosome via RNA polymerase, may have several ribo-somes already moving along it making polypeptide chains. This is known as coupled transcription-translation (Fig. 8.21). This is impossible in higher, eukaryotic cells, because the DNA is inside the nucleus and the ribosomes are outside, in the cytoplasm. [Some recent work has suggested that a small amount of translation may occur inside the nucleus of eukaryotes. This issue is presently unresolved. Nonetheless, the majority of eukaryotic translation occurs in the cytoplasm, outside the nucleus.]

Ribosomes that have stalled due to defective mRNA can be rescued by a special RNA— tmRNA.

Some Ribosomes Become Stalled and Are Rescued

Cellular metabolism is not perfect and cells must allow for errors. One problem ribo-somes sometimes run across is defective mRNA that lacks a stop codon. Whether synthesis of the mRNA was never completely finished or whether it was mistakenly snipped short by a ribonuclease, problems ensue. In the normal course of events, a ribo-some that is translating a message into protein will, sooner or later, come across a stop codon. Even if an mRNA molecule comes to an abrupt end, ribosomes may be released only by release factor and this in turn needs a stop codon. If the mRNA is defective and there is no stop codon, a ribosome that reaches the end could just sit there forever and the ribosomes behind it will all be stalled, too.

Bacterial cells contain a small RNA molecule that rescues stalled ribosomes. This is named tmRNA because it acts partly like transfer RNA and partly like messenger RNA. Like a tRNA, the tmRNA carries alanine, an amino acid. When it finds a stalled coupled transcription-translation When ribosomes of bacteria start translating an mRNA molecule that is still being transcribed from the DNA tmRNA Specialized RNA used to terminate protein synthesis when a ribosome is stalled by a damaged mRNA

FIGURE 8.22 Stalled Ribosome Liberated by tmRNA

Binding of a tmRNA carrying alanine allows the translation of a damaged message to continue. First alanine is added, then a short sequence of about 10 amino acids encoded by the tmRNA. Finally the stop codon of the tmRNA allows proper termination of the polypeptide chain.

Eukaryotic ribosomes are larger than those of prokaryotes.

ribosome, it binds beside the defective mRNA (Fig. 8.22). Protein synthesis now continues, first using the alanine carried by tmRNA, and then continuing on to translate the short stretch of message that is also part of the tmRNA. Finally, the tmRNA provides a proper stop codon so that release factor can disassemble the ribosome and free it for it for continued protein synthesis. The tRNA domain of tmRNA lacks an anti-codon loop and a D-loop. A protein known as SmpB (not shown in Fig. 8.22 for clarity) binds to the tRNA domain and makes contacts to the ribosome that would normally be made by the missing D-loop.

Clearly, the protein that has just been made is defective and should be degraded. As might be supposed, the tmRNA has signaled that the protein that was made is defective. The short stretch of 11 amino acids specified by the message part of tmRNA and added to the end of the defective protein acts as a signal, known as the ssrA tag. [SsrA stands for small stable RNA A, a name used for tmRNA before its function was elucidated.] The ssrA tag is recognized by several proteases (originally referred to as "tail specific protease") which degrade all proteins carrying this signal. These include the Clp proteases and the HflB protease involved in the heat shock response (see Ch. 9). Eukaryotic cells lack tmRNA. Since they do encounter stalled ribosomes, they presumably have some presently unknown mechanism to deal with this situation.

Differences between Eukaryotic and Prokaryotic Protein Synthesis

The overall scheme of protein synthesis is similar in all living cells. However, there are significant differences between bacteria and eukaryotes. These are summarized in Table 8.02 and discussed in the following sections. Note that eukaryotic cells contain mitochondria and chloroplasts, which have their own DNA and their own ribosomes. The ribosomes of these organelles operate similarly to those of bacteria and will be considered separately below. In eukaryotic protein synthesis, it is usually the cyto-plasmic ribosomes that translate nuclear genes. Several aspects of eukaryotic protein synthesis are more complex. The ribosomes of eukaryotic cells are larger and contain more rRNA and protein molecules than those of prokaryotes. In addition, eukaryotes have more initiation factors and a more complex initiation procedure.

A few aspects of protein synthesis are actually less complex in eukaryotes. In prokaryotes, mRNA is polycistronic and may carry several genes that are translated to give several proteins. In eukaryotes, each mRNA is monocistronic and carries only a single gene, which is translated into a single protein. In prokaryotes, the genome and the ribosomes are both in the cytoplasm, whereas in eukaryotes the genome is in the nucleus. Consequently, coupled transcription and translation is not possible for eukaryotes (except for their organelles; see below).

Both prokaryotes and eukaryotes have a special initiator tRNA that recognizes the start codon and inserts methionine as the first amino acid. In prokaryotes, this first methionine has a formyl group on its amino group (i.e., it is N-formyl-methionine) but in eukaryotes unmodified methionine is used.

Eukaryotic mRNA is recognized by its cap structure (not by base pairing to rRNA).

Initiation of Protein Synthesis in Eukaryotes

Initiation of protein synthesis differs significantly between prokaryotes and eukary-otes. Eukaryotic mRNA has no ribosome binding site (RBS). Instead recognition and binding to the ribosome rely on a component that is lacking in prokaryotes. The cap structure, at the 5'-end, is added to eukaryotic mRNA before it leaves the nucleus (see Ch. 12). Cap binding protein (one of the subunits of eIF4) binds to the cap of the mRNA (Fig. 8.23). Eukaryotes also have more initiation factors than prokaryotes and tail specific protease Enzyme that destroys mis-made proteins by degrading them tail first, i.e., from the carboxyl end

Initiation of Protein Synthesis in Eukaryotes 219

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