Ribosomes, continued

Fig. 9-18 The tryptophan operon and its reguiation showing the situation with moderateiy iow concentrations of tryptophan. The terminator structure forms when the ribosome continues transiation past the trp codons in the ieader peptide and biocks region 2. With criti-caiiy iow concentrations of tryptophan, the ribosome staiis at trp codons, favoring formation of a stem ioop structure that permits transcription to continue.

A system of membrane enclosed cisternae in the cytoplasm. The ER is continuous with the outer membrane of the nuclear envelope. The part of the ER coated with ribosomes is called rough ER, the other part is called smooth-surfaced ER. The rough ER is the first compartment of the secretory pathway. Here, membrane proteins are integrated into and secretory proteins translocated across the ER membrane. Furthermore,... 

Mitochondria have their own DNA (mtDNA) and genetic continuity. This DNA only encodes 13 peptide subunits synthesized in the matrix that are components of complexes I, III, IV, and V of the respiratory chain. Most mitochondrial proteins are synthesized on cytoplasmic ribosomes and imported by specific mechanisms to their specific locations in the mitochondrion (see below). 

Collecting the correct fraction is greatly facilitated by the use of a continuous ultraviolet (UV) detector, such as the ISCO UA-6 system. The OD254 reading allows accurate determination of the sedimentation of various complexes (e.g., 40S, 80S, and various polysomal complexes Fig. 9.1, step 3a). This information can be used to correct for small variations in sedimentation. Importantly, in many cases, it enables the determination of the number of ribosomes on the mRNA or on the resulting fragments, thereby allowing more accurate conclusions. 

The er is a three-dimensional membrane system (57-62). As visualized in a transmission electron microscope, there are two parallel membranes with an intervening electron transparent space, the lumen. The form and abundance of the er vary. The rer are flattened sacs with numerous attached ribosomes (15-20 nm). In contrast, the smooth er (ser) lacks ribosomes. The er seems to function as a communication system within cells and can be continuous with the outer nuclear envelope. Although the rer is involved in protein synthesis, the ser functions in glycosylation. 

A ribosome begins to synthesize the leader peptide, but stalls at the histidine codons because it cannot readily find histidine. Because the ribosome is covering up a different part of the mRNA, the message wiU not fold into the correct terminator structure, and RNA polymerase continues transcription through the structural genes of the operon. Translation of the message produces all the enzymes of the histidine biosynthetic pathway. 

The hierarchy of protein structure is illustrated in hgure 11.4. Here too we have a wealth of structural information. The quaternary structures for many proteins are now known and generally available in databases. As complex as these are, this is not the end of the story. We have atom-by-atom structures for entities as complex as viruses and the ribosome, an intracellular RNA-protein complex and the site of protein synthesis. Modem structural biology continues to provide detailed insights into some of the most complex constracts of nature. We are better off for having these insights. 

Then the peptidyl-tRNA at the A site is translocated to the P site by the ribosome moving along the mRNA a codon at a time, exposing the A site for a new aminoacyl-tRNA appropriate for the particular codon, and a repeat of the elongation process occurs. The cycles of elongation and translocation continue until a termination codon is reached, and the peptide or protein is then hydrolysed and released... 

The nucleus is separated from the cytoplasm by the nuclear envelope, which consists of the outer and inner nuclear membranes. Each of the two nuclear membranes has two layers, and the membranes are separated from each other by the perinuclear space. The outer nuclear membrane is continuous with the rough endoplasmic reticulum and is covered with ribosomes. The inner side of the membrane is covered with a protein layer (the nuclear lamina), in which the nuclear structures are anchored. 

We have shown that out of fifteen forms of three-dimensional crystals from ribosomal particles, grown so far in our laboratory, some appear suitable for crystallographic data collection when using synchrotron radiation at temperatures between 19 °C and —180 °C 50S subunits from H. marismortui., and from B. stearothermophilus, including the -BLl 1 mutant, and the new crystal forms from B. stearothermophilus SOS and Thermus thermophilus 30S subunits which have only recently been grown in non-volatile precipitants We also plan to continue research on biochemically modified particles, such as SOS with one tRNA and its nascent polypeptide chain (which have already been crystallized). 

The mRNA of eucaryotes does not possess specific initiation sequences. Rather, the AUG start codon is identified by scanning the eucaryotic mRNA the 408 subunit of the ribosome threads the 5 non-translated end of the mRNA and uses the first AUG codon encoimtered to initiate translation. Whether a AUG codon is used as an initiator depends, additionally, upon the sequence context. If the sequence environment is unfavorable for initiation, then the scanning is continued and initiation occurs at one of the next AUG. With the help of this leaky scanning" strategy it is possible to produce proteins with different N-termini from the same mRNA. 8ince there are often signal sequences foimd at the N-terminus, this mechanism may lead to alternative com-partmentalization of a protein. 

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