Elongation and Termination of the mRNA

Once the RNA polymerase binds to the promoter, transcription begins at position +1. The polymerase reads the DNA in a 3' to 5' direction, while synthesizing the 1° RNA transcript in a 5' to 3' direction. In bacteria, the complete transcript serves as the template for translation. Transcription ends with termination. The mRNA must be terminated in bacteria; termination of the transcript can result from attenuation or the formation of hairpin structures. Termination occurs at several sites beyond the polyadenylation signal in eukaryotic cells and is dependent on bases near the stop codon.15 Because the eukaryotic cell transcripts are polyadenylated, a termination of the transcription process by a process similar to attenuation is not necessary to regulate gene expression. Specifically, transcripts produced after the polyadenylation signal lack a 5' cap, resulting in rapid degradation.16,17

In eukaryotic cells, once the 1° RNA transcript has been produced in the nucleus, this transcript is processed to form an mRNA by splicing to remove the non-protein-coding introns (intervening sequences) and join the protein-coding exons. Introns are located between sequences called exons, which encode the protein sequence and are translated during protein synthesis. Splicing involves a complex of ribonucleoproteins known as a spliceosome, which recognizes consensus sequences at the 5' and 3' ends of the intron. Genetic changes to these splice donor (A/C AG G U A/G AGU) and splice acceptor ([U/C] 11 N C/U AG G/A) consensus sequences may prevent the spliceosome from recognizing and catalyzing the splicing event.18,19 Autoantibodies directed to or alterations in the steady-state level of the spliceosome may play a role in some diseases.20-22 Alternate splicing may generate multiple distinct transcripts from a single gene. That is, some exons may be spliced out in one mRNA molecule but retained in another. As a result, alternate splicing generates different proteins from the same gene and 1° RNA transcript.23-24

An additional mechanism of generating diversity from 1° RNA transcripts entails trans-splicing (initially identified in Drosophila cells). Essentially, two separate, unrelated transcripts form a hybrid molecule by using the splice donor from the first transcripts and the splice acceptor from the second transcripts. Complementary intronic sequences in both transcripts facilitate the generation of the chimeric mRNA. This process has not been demonstrated in other eukaryotic cells. However, when the process is used for gene therapy applications, normal gene function has been restored from defective genes using trans-splicing.25-26 Other therapeutic applications for catalytic RNA molecules involve innovative treatments for HIV-infected patients. In this application, synthetic ribozymes cleave drug-resistant variants of HIV.25,27-29

Additional modifications of the 1° RNA eukaryotic transcript enhance the stability and transport of the mRNA. One such modification occurs immediately on the generation of the 1° transcript and involves addition of a 7-methyl guanosine linked in an unusual 5' to 5' linkage to the triphosphate at the 5' end of the transcript, also known as the 5' cap. This cap protects the transcript from degradation. Another 1° transcript modification is cleavage at a polyadenylation signal (AAUAA) near the 3' end of the transcript, followed by the addition of 100 to 200 adenosine residues (poly-A tail) by polyadenylate polymerase. The poly-A tail facilitates transportation of the mature mRNA into the cytoplasm and protection of the transcript from degradation by exonucleases. A given gene may have several polyadenylation signals, providing another level of variation for a single gene.30-32

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