Polyadenylate polymerase

Polarized light interaction with matter, OPTICAL ROTATION Polyadenylate polymerase,... 

Addition of a poly-A tail Most eukaryotic mRNAs (with several notable exceptions, including those coding for the histones and some interferons) have a chain of 40 to 200 adenine nucleotides attached to the 3 -end (see Rgure 30.17). This poly-A tail is not transcribed from the DNA, but rather is added after transcription by the nuclear enzyme, polyadenylate polymerase. A consensus sequence, called the polyadenylation signal sequence (AAUAAA), found near the 3 -end of the RNA molecule, signals that a poly-A tail is to be added to the mRNA. These tails help stabilize the mRNAs and facilitate their exit from the nucleus. After the mRNA enters the cytosol, the poly-A tail is gradually shortened. 

Polyadenylate polymerases are the enzymes that synthesize poly(A) tails usually found at the 3 end of nonhistone eukaryotic mRNAs. Like the poly(A) tailing which occurs posttranscriptionally in the nucleus, the enzyme performs template-independent polyadenylation in vitro at the 3 terminus of a wide variety of RNAs. Its specificity for ATP as an almost exclusive nucleotide substrate, virtual nonspecificity (in the absence of a specificity factor) for acceptor (or initiator) RNA substrates, and distributive mode of poly (A) elongation make the enzyme a useful tool in recombinant DNA technology. The poly(A) polymerase is particularly useful in converting poly(A) RNAs to poly(A) RNAs which can then be reverse transcribed to cDNAs using oligo(dT) as the primer. 

Figure 20.20 Summary of transcription, RNA processing and polypeptide synthesis. Polymerisation of the DNA template by RNA polymerase produces pre-mRNA (the primary transcript) this is transcription. The pre-mRNA is now processed, which involves capping, polyadenylation, editing and splicing (see text). The resultant mRNA transfers from the nucleus to the cytosol, where amino acids are polymerised to produce a polypeptide using the instructions present in the codons of the mRNA.

Cohen presented a method for determining the rate constants for irreversible polymerization where the different rate constants apply to the initial step and the propagation reactions. Data are obtained in the usual manner at moderate concentrations of primer. The approach is equally valid for template-directed and template-independent polymerization. Cohen applied this method to obtain rate constants for polyadenylate [(poly(A)] polymerase (EC 2.7.7.19). The report also contains useful information about simulating the time course of irreversible polymerization. 

In vitro experiments show that correct modification of the 3 -end requires at least three protein factors the CPSF protein, the poly-A polymerase and the poly-A binding protein. The CPSF protein (CPSF cleavage and polyadenylation specificity factor) binds to the AAUAA signal and brings the poly-A polymerase to the polyadenylation site. The poly-A polymerase is supported by the poly-A binding protein. The latter binds to the poly-A sequence and is required for the transition from the phase of synthesis of short poly-A sequences to the formation of mature poly-A sequences (ca. 200 A-residues). 

Skaar, D.A. Greenleaf, A.L. The RNA polymerase II CTD kinase CTDK-I affects pre-mRNA 3 -cleavage/polyadenylation through the processing component Ptilp. Mol. Cell., 10, 1429-1439 (2002)... 

A poly(A) "tail" consisting of -250 residues of adenylic acid is added next by poly(A) polymerase, a component of an enzyme complex that also cleaves the RNA chains.545 57111 Most eukaryotic mRNA is polyadenylated with the exception of that encoding histones. The function of the poly(A) is unclear. It is needed for transport of mRNA out of the nucleus, but it does confer a greatly increased stability to the mRNA in the cytoplasm where the adenylate irnits are gradually removed.307 308 In contrast, in chloroplasts and plant mitochondria polyadenylation is required for rapid degradation of mRNA.571c d Polyadenylation may also increase the efficiency of translation.572 Polyadenylation occurs rapidly within -1 min after transcription is completed. 

The first step in the replication of influenza viruses, which takes place in the cytoplasm, is the synthesis of (+) strands that can serve both as mRNA for synthesis of proteins and as templates for synthesis of new (-) strands. Three of the capsid proteins form the required RNA polymerase. This "transcriptase" is primed preferentially by 5 -capped 10- to 13-nucleotide segments of RNA that have been cut by a viral nuclease from host mRNAs.700 The mRNAs made from viral RNA are polyadenylated and are translated by the host cell s ribosomes. However, some transcripts are used as templates to form viral (-) strands, which... 

Most pre-mRNA transcripts are cleaved post-transcriptionally near the 3 end between a polyadenylation signal (5 -AAUAAA-3 ) and 5 -YA-3 (where Y = a pyrimidine). A GU-rich sequence may also be located further downstream. Specific proteins bind to these sequence elements to form a complex. One of the bound proteins, poly(A) polymerase, then adds a poly(A) tail of up to 250 A residues to the new 3 end of the RNA molecule and poly(A) binding protein molecules bind to this. The poly(A) tail protects the 3 end of the final mRNA against nuclease degradation and also increases translational efficiency of the mRNA. Some pre-mRNAs (e.g. histone pre-mRNAs) are cleaved near the 3 end but no poly(A) tail is added. 

To demonstrate polymerase activity in a model cell, Chakrabarti et al. [79] encapsulated polynucleotide phosphorylase in vesicles composed of dimyris-toylphosphatidylcholine (DMPC). This enzyme can produce RNA from nucleoside diphosphates such as adenosine diphosphate (ADP) and does not require a template, so it has proven useful for initial studies of encapsulated polymerase activity (Fig. 10a). Furthermore, DMPC liposomes are sufficiently permeable so that 5-10 ADP molecules per second enter each vesicle. Under these conditions, measurable amounts of RNA in the form of polyadenylic acid were synthesized and accumulated in the vesicles after several days incubation. The enzyme-catalyzed reaction could be carried out in the presence of a protease external to the membrane, demonstrating that the vesicle membrane protected the encapsulated enzyme from hydrolytic degradation. Similar behavior has been observed with monocarboxylic acid vesicles [80], and it follows that complex phospholipids are not required for an encapsulated polymerase system to function. 

Polyadenylation results in the addition of a poly (A) tail of 40-200 residues at the 3 end of the transcript. The enzyme responsible for this addition is poly (A) polymerase. The function of the poIy(A) tail is unknown. 

Poly A Sepharose Polyadenylic acid mRNA-binding proteins Viral RNA, RNA polymerase Pharmacia... 

Matis, S., Xu, Y., Shah, M Guan, X., Einstein, J. R., Mural, R. Uberbacher, E. (1996). Detection of RNA polymerase II promoters and polyadenylation sites in human DNA sequence. Comput Chem 20,135-40. 

Polyadenylation occurs at the 3 end of the pre-mRNA. First, the pre-mRNA is cleaved when a specific sequence, AAUAAA, is present in the transcript. Cleavage of the pre-mRNA occurs about 20 or so nucleotides downstream (3 ) of the polyA signal sequence. RNA polymerase II continues on the template, sometimes for as long as... 

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