Core enzyme

Figure Bl.9.9. Comparison of the distance distribution fiinction p(r) of a RNA-polymerase core enzyme from the experimental data (open circle) and the simulation data (using two different models). This figure is duplicated from [27], with pennission from Elsevier Science.

Fig. 2. Modulation of the RseA activity a working model. In the absence of non-native proteins in the periplasm, is bound to the RseA anti-sigma factor this binding is stabilized by RseB, a co-anti-sigma factor, in the periplasm. Upon accumulation of non-native proteins within the periplasm, RseB is released followed by dissociation of which interacts with RNA polymerase core enzyme to initiate transcription of the heat shock genes of the sigma-E regulon...

Primary structure analysis of phenylphosphate carboxylase of T. aromatica is performed in detail, to clarify the reaction mechanism involving four kinds of subunits. The a, (3, y, 8 subunits have molecular masses of 54, 53, 18, and lOkDa, respectively, which make up the active phenylphosphate carboxylase. The primary structures of a and (3 subunits show homology with 3-octaprenyl-4-hydroxybenzoate decarboxylase, 4-hydroxybenzoate decarboxylase, and vanil-late decarboxylase, whereas y subunit is unique and not characterized. The 18kDa 8 subunit belongs to a hydratase/phosphatase protein family. Taking 4-hydroxybenzoate decarboxylase into consideration, Schiihle and Fuchs postulate that the a(3y core enzyme catalyzes the reversible carboxylation. ... 

Colland, F., Fujita, N., Ishihama, A., and Kolb, A. (2002) The interaction between sigmaS, the stationary phase sigma factor, and the core enzyme of Escherichia coli RNA polymerase. Genes Cells 7, 233-247. 

RNA polymerase Core enzyme RNA polymerase I rRNA RNA polymerase II mRNA snRNA RNA polymerase III tRNA, 5S RNA... 

The core-enzymes, prepared in our laboratory, and containing the active centers, were successfully crystallized (Dr. Jones, Uppsala, communicated) and tertiary structures will be described in the near future. Chemical modification studies on these enzymes are currently being undertaken in our laboratory identification of important catalytic residues and location of the active centers will lead to more functional information on these enzymes. Other cellulases such as some endoglucanases from Clostridium thermocel-lum (EG A, EG B, EG D) (10) and EngA and Exg from Cellulomonas fimi (19) also contain sequences of conserved, terminally located and sometimes reiterated, amino acids. Some of these sequences are preceded by proline-serine rich domains. Thus, a bistructural-bifunctional organization seems to be a rather common feature among cellulases, at least for EngA and Exg from C. fimi and the enzymes from Trichoderma reesei. 

The core-enzymes (15,16) showed reduced adsorption capacities on Avicel of more than 50%, whereas the adsorptions on amorphous (H3P04-Swollen) cellulose were unaffected. This emphasized the role of the binding domains as described above. 

The hydrolytic activities of the intact enzymes were comparable, but CBH I was much more sensitive to product (cellobiose) inhibition. Both core enzymes exhibited a strongly reduced activity (50-90%) which was correlated with the absence of the binding domain and their consequent lower binding capacity on Avicel. The activities of CBH I and Core I on amorphous cellulose were, however, comparable. 

The RNA polymerase of E. coli possesses with its subimit construction (a2PP o) a simple structure in comparison to eucaryotic RNA polymerases. The sigma factor is only required for the recognition of the promoter and the subsequent formation of a tight complex. After the incorporation of the first 8-10 nucleotides into the transcript, the sigma factor dissociates from the holoenzyme, and the remaining core enzyme carries out the rest of the elongation. 

General transcription initiation factors TFIIB, TFIIE, TFIIF and TFIIH have been identified as components of the RNA polymerase 11 holoenzyme of yeast. Various forms of the yeast holoenzyme contain further proteins, known as mediators or SRB proteins (SRB, suppressor of RNA polymerase B). The mediators fimction as coactivators (see 1.4.3.2). The holoenzyme is difficult to define structurally because the proteins accessory to the core enzyme (see table 1) may not be permanently associated with RNA polymerase II. 

Core enzyme Four of the enzyme s peptide subunits, 2a, 1p, and 1p are responsible for the 5 ->3 RNA polymerase activity, and are referred to as the core enzyme (Figure 30.6). However, this enzyme lacks specificity, that is, it cannot recognize the promoter region on the DNA template. 

Holoenzyme The c subunit ( sigma factor") enables RNA polymerase to recognize promoter regions on the DNA. The o subunit plus the core enzyme make up the holoenzyme. [Note Different o factors recognize different groups of genes.]... 

There are three major types of RNA that participate in the process of protein synthesis ribosomal RNA (rRNA), transfer RNA (tRNA), and messenger RNA (mRNA). They are unbranched polymers of nucleotides, but differ from DNA by containing ribose instead of deoxyribose and uracil instead of thymine. rRNA is a component of the ribosomes. tRNA serves as an adaptor molecule that carries a spe dfic amino acid to the site of protein synthesis. mRNA carries genetic information from the nuclear DNA to the cytosol, where it is used as the template for protein synthesis. The process of RNA synthesis is called transcription, and its substrates are ribonucleoside triphosphates. The enzyme that synthesizes RNA is RNA polymerase, which is a multisub-irit enzyme. In prokaryotic cells, the core enzyme has four subunits—... 

The process of RNA synthesis is called transcription. The enzyme that synthesizes RNA is RNA polymerase, which is a multisubunit enzyme. The core enzyme has four subunits—2 a, 1 p, and 1 p, and possesses 5 —>3 polymerase activity. The enzyme requires an additional subunit—sigma (a) factor—that recognizes the nucleotide sequence (promoter region) at the beginning of a length of DNA that is to be transcribed. Another protein—rho (p) factor—is required for termination of transcription of some genes. 

The enzyme without the sigma factor, called core polymerase, retains the capability to synthesize RNA, but it is defective in the ability to bind and initiate transcription at true initiation sites on the DNA. In fact when RNA polymerase was first purified from crude extracts it was missing the a factor. The assay for polymerase involved the use of DNA with single-strand nicks. When a DNA template was used that did not have single-strand nicks, this enzyme was not active. This led to a search for a missing factor. When this factor (cr70) was added back to the purified core enzyme and the uncut DNA template, the enzyme was able to bind... 

The precise functions of the subunits of the core enzyme are not known. /3 is a basic (positively charged) polypeptide thought to be involved in DNA binding. The j8 subunit is the site of binding of several inhibitors of transcription and is thought to contain most or all of the active sites for phosphodiester bond formation. The a subunit is necessary for reconstituting active enzyme from separated subunits. 

DNA-dependent synthesis of RNA in E. coli is catalyzed by one enzyme, consisting of five polypeptide subunits. The complete holoenzyme is composed of four polypeptides (the core enzyme) and an additional polypeptide that confers specificity for initiation at promoter sequences in the DNA template. 

Following initiation, the a subunit dissociates from RNA polymerase to leave the core enzyme ( 2PP m) that continues RNA synthesis in a 5 — 3 direction using the four ribonucleoside 5 triphosphates as precursors. The DNA double helix is unwound for transcription, forming a transcription bubble, and is then rewound after the transcription complex has passed. 

In E. coli, all genes are transcribed by a single large RNA polymerase with the subunit structure a2pp a. This complete enzyme, called the holoenzyme, is needed to initiate transcription since the a factor is essential for recognition of the promoter it decreases the affinity of the core enzyme for nonspecific DNA binding sites and increases its affinity for the promoter. It is common for prokaryotes to have several a factors that recognize different types of promoter (in E. coli, the most common a factor is a70). 

Each of the three eukaryotic RNA polymerases contains 12 or more subunits and so these are large complex enzymes. The genes encoding some of the subunits of each eukaryotic enzyme show DNA sequence similarities to genes encoding subunits of the core enzyme (a2PP ) of E. coli RNA polymerase (see Topic G2). However, four to seven other subunits of each eukaryotic RNA polymerase are unique in that they show no similarity either with bacterial RNA polymerase subunits or with the subunits of other eukaryotic RNA polymerases. 

When isolated from bacteria, prokaryotic RNA polymerase has two forms The core enzyme and the holoenzyme. The core enzyme is a tetramer whose composition is given as 0C2PP (two alpha subunits, one beta subunit, and one beta-prime subunit). Core RNA polymerase is capable of faithfully copying DNA into RNA but does not initiate at the correct site in a gene. That is, it does not recognize the promoter specifically. Correct promoter recognition is the function of the holoenzyme form of RNA polymerase. 

The initiator nucleotide binds to the complex and the first phos-phodiester bonds are made, accompanied by release of o. The remaining core polymerase is now in the elongation mode. Several experimental observations support the picture presented in the next figure, namely the fact that less than one a exists in the cell per core enzyme in each cell. 

Elongation is the function of the RNA polymerase core enzyme. RNA polymerase moves along the template, locally unzipping the DNA double helix. This allows a transient base pairing between the incoming nucleotide and newly-synthesized RNA and the DNA template strand. As it is made, the RNA transcript forms secondary structure... 

While the CHS-CHI-F3H-DFR-AS enzymes form the core flavonoid biosynthetic pathway (Fig. 3.2), every intermediate compound in the pathway can be the subject of complex modifications that include hydroxylations, methylations, esterifications, and decorations with a number of sugar moieties. In addition, many of the core enzymes can utilize various substrates resulting in a pathway that is not linear, but rather a complex grid (Fig. 3.2).2 The diverse forms of flavonoids or anthocyanins that accumulate in any plant under any given condition are the result of a combination of the biosynthetic enzymes being expressed together with their substrate specificity. Over the past few years, the structures of several flavonoid biosynthetic enzymes have been elucidated,1 -20 which opens up unlimited opportunities to understand structure-function relationships and to manipulate the pathway. 

In maize, all the genes encoding the core enzymes of the flavonoid pathway (Fig. 3.2) are regulated by a combination of the R2R3 MYB factors Cl or PL and the... 

DNA polymerase III is also a multifunctional enzyme. It resembles DNA polymerase I in catalytic properties however, there are slight differences with respect to the type of template primer preferred for DNA synthesis as well as the preferred substrates for the two exonuclease activities. It contains many polypeptide subunits (a, e, 6, t, y, 8. y, ip and B)- The complex containing all subunits (Mr 900,000) is called the DNA polymerase III holoenzyme, while that comprising just a, e and 8 exhibits the polymerase activity and is referred to as the core enzyme. The holoenzyme carries out most of the DNA synthesis at the replication fork in vivo. 

What constitutes the DNA polymerase III core enzyme of E. coli ... 

DNA polymerase III occurs within the cell as a functional complex of 10 polypeptide chains. This is called the holoenzyme. A subcomplex containing three of these chains (a, e, and 6) is readily isolated and exhibits polymerase activity. It is called the DNA polymerase III core enzyme. 

---