Eukaryotic mRNA production

In some circumstances the aim of the experiment will be to clone a DNA sequence which lacks cohesive ends. Such molecules may, for example, be the cDNA products from the reverse transcription of particular mRNA species and this is currently the most widely used procedure for sequencing eukaryotic mRNAs. Under controlled conditions the reverse transcriptase from Avianmyeloblas-tosis virus (AMV) will use a mRNA template to synthesize a full-length double-stranded DNA copy (cDNA) of the RNA sequence. The products of this reaction frequently lack precisely defined ends and it is necessary to trim such frayed or uneven termini before proceeding to the cloning step. Incubation with DNA polymerase (Klenow) or T4-polymerase (Section 4.2.2.) will yield molecules with clean blunt-ended termini which can be... 

Schematic drawing showing production of eukaryotic mRNA. The primary transcript is capped before it is released. Then its 3 -OH end is modified, and finally the intervening regions are excised. MeG, 7-methylguanosine. Two nucleotides whose riboses may be methylated.

The answer is c. (Murray, pp 452-467. Scriver, pp 3-45. Sack, pp 1-40. Wilson, pp 101-120.) In a general sense, the mechanism of protein synthesis in eukaryotic cells is similar to that found in prokaryotes however, there are significant differences. Cycloheximide inhibits elongation of proteins in eukaryotes, while erythromycin causes the same effect in prokaryotes. Thus, one is an antibiotic beneficial to humans, while the other is a poison. Cytoplasmic ribosomes of eukaryotes are larger, sedimenting at SOS instead of 70S. While eukaryotic cells utilize a specific tRNA for initiation, it is not formylated as in bacteria. Finally, eukaryotic mRNA always specifies only one polypeptide, as opposed to prokaryotic mRNA, which may specify the synthesis of more than one gene product per mRNA. 

Eukaryotes - In eukaryotes, the mRNA is produced in the nucleus and must be exported into the cytosol for translation. Furthermore, the initial product of transcription (pre-mRNA) may include introns, which must be removed before translation can occur. There is no ribosomal attachment sequence like the Shine - Dalgamo sequence in prokaryotes. For all these reasons, eukaryotic mRNA requires extensive processing before it can be used as a protein template. This processing takes place while mRNA is still in the nucleus. 

Polyadenylation is a critical posttranscriptional processing step in the maturation of eukaryotic mRNA [1], The location where the pre-mRNA is cleaved (also known as the poly(A) site) marks the end of a mRNA transcript. Many eukaryotic genes possess two or more poly(A) sites [2-4], and thus are involved in alternative polyadenylation (APA). APA is a powerful pathway that entails the selection of alternate poly(A) sites in a pre-mRNA and leads to the production of multiple mature mRNA isoforms from the same gene, resulting in potential gene expression regulation [1]. 

The eukaryotic expression cassette is the part of an expression vector that enables production of a protein in a eukaryotic cell. The cassette consists of a eukaryotic promoter for mRNA transcription, the gene and an mRNA termination and processing signal (Poly-A signal). 

Figure 37-1. This figure illustrates that genes can be transcribed off both strands of DNA. The arrowheads indicate the direction of transcription (polarity). Note that the template strand is always read in the 3 to 5 direction. The opposite strand is called the coding strand because it is identical (except for T for L) changes) to the mRNA transcript (the primary transcript in eukaryotic cells) that encodes the protein product of the gene.

The relationship between hnRNA and the corresponding mature mRNA in eukaryotic cells is now apparent. The hnRNA molecules are the primary transcripts plus their early processed products, which, after the addition of caps and poly(A) tails and removal of the portion corresponding to the introns, are transported to the cytoplasm as mature mRNA molecules. 

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