Polyadenylation poly

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. 

As mentioned earlier, pre-mRNA is also modified at the 3 end. Most eukaryotic mRNAs contain a polyadenylate, poly (A), tail at that end, added after transcription has ended. Thus, DNA does not encode this poly(A) tail. Indeed, the nucleotide preceding poly(A) is not the last nucleotide to be transcribed. Some primary transcripts contain hundreds of... 

When 2, 3 -cyclic nucleotides are treated with tetraphenylpyrophos-phate at room temperature a rapid exothermic polymerization takes place to yield an unnatural polynucleotide with mixed 2, 5 - and 3, 5 -inter-nucleotide linkages and a terminal 2, 3 -cyclic phosphate. Each polymeric molecule contains two to ten nucleotide residues. Polyadenylic, poly-uridylic, and mixed polynucleotides have been made in this manner 216) [Eq. 56]. 

Figure 39-19. Structure of a typical eukaryotic mRNA showing elements that are involved in regulating mRNA stability. The typical eukaryotic mRNA has a 5 noncoding sequence (5 NCS), a coding region, and a 3 NCS. All are capped at the 5 end, and most have a polyadenylate sequence at the 3 end. The 5 cap and 3 poly(A) tail protect the mRNA against exonuclease attack. Stem-loop structures in the 5 and 3 NCS, features in the coding sequence, and the AU-rich region in the 3 NCS are thought to play roles in mRNA stability.

Figure 1. Expression of c-mos in mouse oocytes, c-mos is transcribed during oocyte growth and transcripts with short poly(A) tails are accumulated in fully-grown germinal vesicle (GV) stage oocytes. These transcripts are polyadenylated and translated following the resumption of meiosis and then degraded following fertilization and cleavage to the two-cell stage.

At this stage, mRNA can be polyadenylated using the poly(A) tailing kit (Ambion), according to the manufacturer s instructions. 

Example experiments using the previous methodologies are shown in Fig. 6.1. The major mRNA constructs described in this chapter are dia-grammatically represented in Fig. 6. IB and an example of in vitro transcribed and polyadenylated R-luc-4 sites mRNA is shown in Fig. 6.1A. In these experiments, translation of R-luc-4 sites mRNA is synergistically promoted by the physiological cap structure and the poly (A) tail (Fig. 6.1C), and full miR-dependent translational repression requires the presence of both modifications (Fig. 6.ID, Humphreys etal., 2005). (TheEMCV IRES-containing constructs are discussed later.)... 

Polyadenylation This process is the addition of many AMP molecules to the 3 end of the RNA molecule. Once bound it is known as a poly A tail, since it is comprised of 100 or more such adenylate (AMP) residues. The tail is thought to stabilise the mRNA molecule in the cytosol, probably by preventing hydrolysis by an RNAase. 

In higher eucaryotes the 3 -end of matme mRNA is not produced as a result of termination of transcription. Rather, the 3 -end of the primary transcript is cut at a specific site and a poly-A sequence is appended. Polyadenylation precedes the splicing of the primary transcript. 

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). 

FIGURE 26-19 Two mechanisms for the alternative processing of complex transcripts in eukaryotes, (a) Alternative cleavage and polyadenylation patterns. Two poly(A) sites, A, and A2, are shown. 

Fukaryotic mRNAs are modified by addition of a 7-methylguanosine residue at the 5 end and by cleavage and polyadenylation at the 3 end to form a long poly(A) tail. 

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. 

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. 

Table 5.24. 13C Chemical Shifts (6C in ppm) of Randomly Coiled Poly(8-bromoadenylic Acid), Polyadenylic Acid and Corresponding 5 -Mononucleotides Temperature 70 °C Solvent D2Q, pD 7.0-7.3 [781],...

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