Nucleotide sequencing steps

As the second educt (B), the plasmid ONA with complementary sticky ends is prepared separately. In the first step the isolated plasmid DNA is cut open by a special type of enzyme called restriction endonuclease. It scans along the thread of DNA and recognizes short nucleotide sequences, e.g., CTGCAG, which ate cleaved at a specific site, e.g., between A and G. Some 50 of such enzymes are known and many are commercially available. The ends are then again extended witfa he aid of a terminal transferase by a short sequence of identical nucleotides complementary to the sticky ends of educt (A). 

Combinatorial Hbraries are limited by the number of sequences that can be synthesized. For example, a Hbrary consisting of one molecule each of a 60-nucleotide sequence randomized at each position, would have a mass of >10 g, weU beyond the capacity for synthesis and manipulation. Thus, even if nucleotide addition is random at all the steps during synthesis of the oligonucleotide only a minority of the sequences can be present in the output from a laboratory-scale chemical DNA synthesis reaction. In analyzing these random but incomplete Hbraries, the protocol is efficient enough to allow selection of aptamers of lowest dissociation constants (K ) from the mixture after a small number of repetitive selection and amplification cycles. Once a smaller population of oligonucleotides is amplified, the aptamer sequences can be used as the basis for constmcting a less complex Hbrary for further selection. 

An essential step of NR action is the interaction of these receptors with the specific DNA sequence HREs. Indeed, HREs position the receptors, and the transcriptional complexes recruited by them, close to the target genes. HREs are bipartite elements that are composed of two hexameric core half-site motifs. These consensus nucleotide sequences form direct, indirect, or inverted repeats, which consist of two half-sites separated... 

The first step in this process involves the binding of GTP by eIF-2. This binary complex then binds to met-tRNAf a tRNA specifically involved in binding to the initiation codon AUG. (There are two tRNAs for methionine. One specifies methionine for the initiator codon, the other for internal methionines. Each has a unique nucleotide sequence.) This ternary complex binds to the 40S ribosomal subunit to form the 43S preinitiation complex, which is stabilized by association with eIF-3 and elF-lA. 

Various verification steps have been introduced to ensure that SPTR is comprehensive and contains all relevant data sources. The main source of new protein sequences is the translations of CDS in the nucleotide sequence databases. The up-to-date inclusion of new protein sequence entries is ensured by the weekly translation of EMBL-NEW (the updates to the EMBL nucleotide sequence database). The three collaborating nucleotide sequence databases DDBJ, EMBL, and GenBank exchange their data on a daily basis. Therefore any protein coding sequence submitted to DDBJ/EMBL/GenBank will appear in SPTR within 2 weeks in the worst case and within less than 1 week in the average case. 

Interpretation of the electron density maps showed that the large subunit could not be modelled beyond His536 (Fig. 6.10), that is fifteen amino acids short of the 551 residues predicted by the nucleotide sequence (Table 6.2). At about the same time, the cleavage of this fifteen-residue stretch, which is performed by a specific protease, was reported to be an obligatory step for the maturation of the enzyme (Menon et al. 1993). It is also of interest to note that in all [NiFe] hydrogenase crystal structures this buried C-terminal histidine is ligated to a metal atom which is either a magnesium or an iron (see above). 

The transfer of genetic information from the level of the nucleic acid sequence of a gene to the level of the amino acid sequence of a protein or to the nucleotide sequence of RNA is termed gene expression. The entire process of gene expression in eucaryotes includes the following steps ... 

The pathway of protein synthesis translates the three-letter alphabet of nucleotide sequences on mRNA into the twenty-letter alphabet of amino acids that constitute proteins. The mRNA is translated from its 5 -end to its 3 -end, producing a protein synthesized from its amino-terminal end to its carboxyl-terminal end. Prokaryotic mRNAs often have several coding regions, that is, they are polycistronic (see p. 420). Each coding region has its own initiation codon and produces a separate species of polypeptide. In contrast, each eukaryotic mRNA codes for only one polypeptide chain, that is, it is monocistronic. The process of translation is divided into three separate steps initiation, elongation, and termination. The polypeptide chains produced may be modified by posttranslational modification. Eukaryotic protein synthesis resembles that of prokaryotes in most details. [Note Individual differences are mentioned in the text.]... 

In the next step, translation, the sequence of nucleotides in the newly synthesized mRNA strand is used to determine the sequence of amino acids in the protein to be synthesized. This is done by way of a genetic code, which was fully deciphered by 1966 and is shown in Figure 13.34. According to the genetic code, it takes three mRNA nucleotides—each three-nucleotide unit is called a codon—to code for a single amino acid. The mRNA nucleotide sequence AGU, for example, codes for the amino acid serine, and AAG codes for lysine. (Note from Figure 13.34 that more than one codon can call for the same amino acid.) A few codons, such as AUG and UGA, are the signals for protein synthesis to either start or stop. 

Among the new RNA structures are those of RNA-antisense RNA pairs in "kissing" hairpin complexes 87,319 Another interesting complex folding pattern in RNA is the pseudoknot, a structural feature that has been identified in many RNA sequences.85,320 327 A pseudoknot can be formed if nucleotide sequences favorable to formation of two short RNA stems are overlapped as shown in Fig. 5-29. After stem 2 in this drawing is formed (step a) additional base pairing can lead to formation of stem 1 (step b). The base pairs of the two stems can stack coaxially to form the pseudoknot (step c). 

TRANSLATION The process by which a particular messenger RNA (mRNA) nucleotide sequence determines a specific amino acid sequence of a polypeptide chain occurs as the polypeptide is synthesized and is therefore the second step in the readout of the information in the genetic code (the first is transcription). 

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