Poly-A polymerase

Poly(A) tails are added to the S end of mRNA molecules in a posttranscriptional processing step. The mRNA is first cleaved about 20 nucleotides downstream from an AAUAA recognition sequence. Another enzyme, poly(A) polymerase, adds a poly(A) tail which is subsequently extended to as many as 200 A residues. The poly(A) tail appears to protect the S end of mRNA from S —> S exonuclease attack. The presence or absence of the poly(A) tail does not determine whether a precursor molecule in the nucleus appears in the cytoplasm, because all poly(A)-tailed hnRNA molecules do not contribute to cytoplasmic mRNA, nor do all cytoplasmic mRNA molecules contain poly(A) tails... 

Variations The poly(A) tailing kit (Ambion) produces a mRNA population with varying lengths of poly(A) tails, controlled by altering poly(A) polymerase concentrations and incubation times. An alternate method to incorporate a poly(A) tail is to clone a defined stretch of adenosines/ thymidines into the > UTR of the template pDNA. To allow transcripts to finish on an adenosine, the insert should be followed by a restriction site for an enzyme that cleaves 5 of the last antisense strand thymidine, such as Nsi I. In this way, the poly (A) tail can be incorporated directly into the... 

A poly-A tail is attached to the 3 end. In this process, an endonuclease cuts the molecule on the 3 side of the sequence AAUAAA (poly-A addition signal), then poly-A polymerase adds the poly-A tail (about 200 As) to the new 3 end. The poly-A tail protects the message against rapid degradation and aids in its transport to the cytoplasm. A few mRNAs (for example, histone mRNAs) have no poly-A tails. 

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. 

IRREVERSIBLE POLYMERIZATION POLY(ADP-RIBOSE) SYNTHETASE Poly(A) polymerase,... 

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). 

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. 

In eukaryotes, 100-200 adenosine residues are added to the 3 ends of most mRNAs by a poly (A) polymerase. This addition occurs in the nucleus before the mRNA is fully processed and transported to the cytoplasm. 

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. 

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. 

Figure 28.25. Polyadenylation of a Primary Transcript. A specific endonuclease cleaves the RNA downstream of AAUAAA. Poly(A) polymerase then adds about 250 adenylate residues.

J. Bard, A.M. Zhelkovsky, S. Helmling, T.N. Earnest, C.L. Moore, and A. Bohm. 2000. Structure of yeast poly(A) polymerase alone and in complex with 3 -dATP Science 289 1346-1349. (PubMed)... 

G. Martin, W. Keller, and S. Doublie. 2000. Crystal structure of mammalian poly(A) polymerase in complex with an analog of ATP EMBOJ. 19 4193-4203. (PubMed)... 

Polyadenylatiotf. is the addition of a chain of adenylate residues, known as a poly A tail to the 3 terminus of mRNA (Fig 11.12). After the RNA is cut, an enzyme poly A polymerase, catalyzes the polymerization of adenylates. The poly A tail slows the exonucleolytic degradation of mRNA, once the tail is removed mRNA is quickly degraded. 

Adenosine triphosphate, when oxidized to its dialdehyde with periodate and condensed with Sepharose-adipic (or sebacic) acid hydrazide, has been used to isolate poly(A) polymerase, and also myosin and its fragments from different sources. A poly(A)-linked resin has been prepared by condensing 4,4 -diaminodiphenyl-methane with periodate-oxidized starch, reducing the resulting Schiff bases with borohydride, diazotizing, and then coupling with the polynucleotide. The resulting material may be used to isolate poly(U) sequences. 

The nonessential amino acid, L-alanine, has been reported to compete with tryptophan binding to hepatic nuclei in vitro.174485 However, L-alanine is not capable of stimulating hepatic protein synthesis as does L-tryptophan.185 Yet, L-alanine in competing with L-tryptophan for nuclear receptor binding is able to diminish or negate L-tryptophan s ability to stimulate hepatic protein synthesis.185 Similarly, DL- 3(l-naphthyl)alanine is capable of acting like L-alanine.173 Also, L-alanine inhibited elevations of other rapidly induced metabolic reactions, such as nuclear RNA efflux and nuclear poly(A)polymerase activity, which occurred due to L-tryptophan alone. These two stimulatory... 

Kurl, R. N., Holmes, S. C., Verney, E., and Sidransky H., Nuclear envelope glycoprotein with poly(A) polymerase activity of rat liver Isolation, characterization, and immunohistochemical localization, Biochemistry 27[25], 8974,1988. 

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