The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis

抗原变异可使得多种致病微生物易于逃避宿主免疫应答。表达在感染红细胞表面的恶性疟原虫红细胞表面蛋白1（PfPMP1）与感染红细胞、内皮细胞、树突状细胞以及胎盘的单个或多个受体作用，在黏附及免疫逃避中起关键的作用。每个单倍体基因组var基因家族编码约60种成员，通过启动转录不同的var基因变异体为抗原变异提供了分子基础。

疟原虫var基因转换速率变化导致抗原变异[英]／Paul H, Robert P, Christodoulou Z, et al//Proc Natl Acad Sci U S A

A set of oligonucleotide primers capable of initiating enzymatic amplification (polymerase chain reaction) on a phylogenetically and taxonomically wide range of bacteria is described along with methods for their use and examples. One pair of primers is capable of amplifying nearly full-length 16S ribosomal DNA (rDNA) from many bacterial genera; the additional primers are useful for various exceptional sequences. Methods for purification of amplified material, direct sequencing, cloning, sequencing, and transcription are outlined. An obligate intracellular parasite of bovine erythrocytes, Anaplasma marginale, is used as an example; its 16S rDNA was amplified, cloned, sequenced, and phylogenetically placed. Anaplasmas are related to the genera Rickettsia and Ehrlichia. In addition, 16S rDNAs from several species were readily amplified from material found in lyophilized ampoules from the American Type Culture Collection. By use of this method, the phylogenetic study of extremely fastidious or highly pathogenic bacterial species can be carried out without the need to culture them. In theory, any gene segment for which polymerase chain reaction primer design is possible can be derived from a readily obtainable lyophilized bacterial culture.

16S ribosomal DNA amplification for phylogenetic study.

We describe a program, tRNAscan-SE, which identifies 99-100% of transfer RNA genes in DNA sequence while giving less than one false positive per 15 gigabases. Two previously described tRNA detection programs are used as fast, first-pass prefilters to identify candidate tRNAs, which are then analyzed by a highly selective tRNA covariance model. This work represents a practical application of RNA covariance models, which are general, probabilistic secondary structure profiles based on stochastic context-free grammars. tRNAscan-SE searches at approximately 30 000 bp/s. Additional extensions to tRNAscan-SE detect unusual tRNA homologues such as selenocysteine tRNAs, tRNA-derived repetitive elements and tRNA pseudogenes.

tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence.

Phylogenetic identification and in situ detection of individual microbial cells without cultivation.

During protein synthesis, mRNA and tRNAs must be moved rapidly through the ribosome while maintaining the translational reading frame. This process is coupled to large- and small-scale conformational rearrangements in the ribosome, mainly in its rRNA. The free energy from peptide-bond formation and GTP hydrolysis is probably used to impose directionality on those movements. We propose that the free energy is coupled to two pawls, namely tRNA and EF-G, which enable two ratchet mechanisms to act separately and sequentially on the two ribosomal subunits.

The ribosome moves: RNA mechanics and translocation

Ribosomal protein from E. coli separates during gel electrophoresis into a large number of components.'-3 A variety of experiments have been done to determine if the apparent complexity might be misleading and be caused by interactions among a smaller number of components. No evidence has been found in favor of this hypothesis.\"' 3-7 The most likely conclusion is that the multiple components seen on gel electrophoresis correspond to different protein molecules in the ribosome population. The possibility remains, however, that the different electrophoretic components are closely related chemically and are only slight modifications or combinations of one or a few primary structures. This view has been favored by success in elucidating the general mechanism of protein synthesis while considering the ribosome a passive structure or template, and by the plausible analogy between ribosome and virus structure. On the other hand, the recent demonstration that specific fractions of ribosomal protein restore in vitro activity to protein depleted particles6-9 and the evidence indicating that peptidyl transferase is an integral part of the 50S ribosome'0'-2 are more consistent with the concept of the ribosome as an enzyme complex composed of functionally specific, chemically distinct proteins. The experiments presented here were designed to distinguish between these alternatives using two approaches. (1) The number of tryptic peptides of the total protein containing sulfur was examined. The relationship of this number to the total calculated number of sulfur-containing residues should reflect the extent to which common sequences occur. (2) Several pure proteins have been isolated and tryptic fingerprints and amino acid compositions compared. Evidence of both types leads to the conclusion that the ribosomal proteins are chemically heterogeneous. Materials and Methods.-Early and late log phase E. coli B cells were purchased from General Biochemicals, Chagrin Falls, Ohio. Cell extracts were prepared by homogenization with glass beads\"3 or with a Hughes pressure cell. Ribosomes and ribosomal subunits were prepared by standard methods.\"' 15 Ribosomal proteins were extracted with 66 per cent acetic acid.'6 Protein concentrations were determined by the Folin method.'7 N-terminal groups were found by the dinitrofluorobenzene (DNFB) method.'8 19 Acrylamide gel electrophoresis and radioautography were done as described by Traut.3 S36 MRE-60021 cells were grown as described previously.3 Cysteine was labeled selectively by including 10 ,g/ml of nonradioactive methionine in the medium. The exclusion of S35 from methionine was confirmed by analysis of acid hydrolysates by paper electrophoresis at pH 1.9 followed by radioautography. Carboxymethyl cellulose was obtained from Calbiochem (Los Angeles) and from W. and R. Balston, Ltd. (England). Performic acid oxidation was carried out according to Nathans1° and carboxymethylation according to Spackman et al.26 Purification of the proteins was achieved on carboxy-

Ribosomal proteins of Escherichia coli. I. Demonstration of different primary structures

A refined model has been developed for the folding of 16S rRNA in the 30S subunit, based on additional constraints obtained from new experimental approaches. One set of constraints comes from hydroxyl radical footprinting of each of the individual 30S ribosomal proteins, using free Fe(2+)-EDTA complex. A second approach uses localized hydroxyl radical cleavage from a single Fe2+ tethered to unique positions on the surface of single proteins in the 30S subunit. This has been carried out for one position on the surface of protein S4, two on S17, and three on S5. Nucleotides in 16S rRNA that are essential for P-site tRNA binding were identified by a modification interference strategy. Ribosomal subunits were partially inactivated by chemical modification at a low level. Active, partially modified subunits were separated from inactive ones by binding 3'-biotinderivatized tRNA to the 30S subunits and captured with streptavidin beads. Essential bases are those that are unmodified in the active population but modified in the total population. The four essential bases, G926, 2mG966, G1338, and G1401 are a subset of those that are protected from modification by P-site tRNA. They are all located in the cleft of our 30S subunit model. The rRNA neighborhood of the acceptor end of tRNA was probed by hydroxyl radical probing from Fe2+ tethered to the 5' end of tRNA via an EDTA linker. Cleavage was detected in domains IV, V, and VI of 23S rRNA, but not in 5S or 16S rRNA. The sites were all found to be near bases that were protected from modification by the CCA end of tRNA in earlier experiments, except for a set of E-site cleavages in domain IV and a set of A-site cleavages in the alpha-sarcin loop of domain VI. In vitro genetics was used to demonstrate a base-pairing interaction between tRNA and 23S rRNA. Mutations were introduced at positions C74 and C75 of tRNA and positions 2252 and 2253 of 23S rRNA. Interaction of the CCA end of tRNA with mutant ribosomes was tested using chemical probing in conjunction with allele-specific primer extension. The interaction occurred only when there was a Watson-Crick pairing relationship between positions 74 of tRNA and 2252 of 23S rRNA. Using a novel chimeric in vitro reconstitution method, it was shown that the peptidyl transferase reaction depends on this same Watson-Crick base pair.

Structure and function of ribosomal RNA.

This system allows convenient purification of large quantities of all of the small subunit ribosomal proteins by overexpression from cloned genes. This not only allows large-scale reconstitution of 30S subunits from individual proteins, but also facilitates protein purification greatly. These proteins can be reconstituted into functional 30S subunits using an ordered assembly protocol based on the in vitro 30S assembly map. Reconstitution of 30S subunits using this system enables mutant or modified proteins, such as Fe(II)-BABE-derivatized proteins, to be incorporated into subunits for studying ribosome structure and function.

[30] In vitro reconstitution of 30s ribosomal subunits using complete set of recombinant proteins

Hydroxyl radical is a useful probe of the accessibility of the sugar moiety of nucleic acids to solvent. Here we compare the accessibility of free and ribosome-bound yeast tRNA(Phe), Escherichia coli tRNA(Phe), and E. coli tRNA(Leu2) to attack by hydroxyl radicals generated from Fe(2+)-EDTA. When bound to the P site of 30S ribosomal subunits, a discrete region, corresponding almost precisely to the anticodon stem-loop, is strongly protected; weaker protection is observed in the 3' strand of the D stem and in the variable loop. The protected nucleotides constitute a well-defined substructure, corresponding to the lower half of the anticodon-D loop coaxial arm of the tRNA crystal structure. This result suggests that the 30S P site contains a pocket that becomes inaccessible to the Fe(2+)-EDTA complex when tRNA is bound, whose minimum dimensions can be inferred from the boundaries of the protected region of tRNA. When bound to the P site of 70S ribosomes, the entire tRNA backbone becomes inaccessible to hydroxyl radicals. Since previous studies have shown that virtually the entire footprint of a P-site tRNA on 16S and 23S rRNAs is mimicked by the extremities of the tRNA (the anticodon stem-loop plus the 3'-terminal aminoacyl-pentanucleotide), protection of the entire tRNA was unexpected. We conclude that protection of the elbow of tRNA is due either to interactions with ribosomal proteins or to enclosure in an inaccessible site formed by association of the two ribosomal subunits.

Harry F. Noller

Papers

The ribosome moves: RNA mechanics and translocation

Ribosomal proteins of Escherichia coli. I. Demonstration of different primary structures

Structure and function of ribosomal RNA.

[30] In vitro reconstitution of 30s ribosomal subunits using complete set of recombinant proteins

Hydroxyl radical cleavage of tRNA in the ribosomal P site