The mRNA’s nucleotide sequence is translated into amino acids according to a set of rules called the genetic code. EF-Tu, in its GTP-bound form, escorts aminoacyl tRNAs to the ribosome and double-checks that the incoming tRNA is a perfect match for an exposed codon.
Because many codons can indicate the same amino acid, mRNA has redundant start codons, and many of its tRNAs can base-pair with multiple codons.
mRNA Structure
The mRNA molecule has a characteristic secondary structure that folds into precisely defined three-dimensional shapes. Four short segments of the mRNA form a cloverleaf that pairs complementarily with complementary sequences in another part of the mRNA molecule, forming an L-shaped structure. This binding between mRNA and tRNA is called hybridization and is essential to protein synthesis.
The precursor mRNA that comes off the DNA template is a polynucleotide with both non-coding regions (introns) and coding regions (exons). Eukaryotic mRNA goes through several post-transcriptional processing steps to make it mature for protein synthesis. First, the non-coding introns are removed by complexes of RNA and protein molecules called Spliceosomes. Next, the 5′ end of the mRNA is added with a 7-methylguanosine cap and a 3′ end with a polyadenylated tail of numerous adenine nucleotides to make it more stable and prevent its degradation.
In addition, a signal sequence at the start of the mRNA is used to set the correct reading frame for protein synthesis. Finally, mRNA is recognized and translated by tRNAs in the ribosome. During translation, tRNAs function as adaptors to convert the mRNA codons into amino acid sequences matched with specific amino acid chains in the ribosome. Aminoacyl-tRNA synthetases then attach the appropriate amino acids to the chain of peptide bonds. The resulting proteins are released from the ribosome and transported to other cells for further functions.
Transcription
The first step of the mRNA manufacturing process is transcription. This enzymatic reaction produces a single-stranded RNA molecule complementary to one of the two DNA templates. This optimized process consistently yielded 3-5 mg/mL of full-length mRNA drug substance with low residual double-stranded DNA.
Once RNA polymerase finishes transcription, a series of proteins assemble on the pre-mRNA molecule. These include the snRNP proteins that remove hairpin helices, package mRNA for transport into the nucleus, and other protein-RNA complexes that recognize specific sequences and mark exon-exon boundaries. A cap-binding protein adds a modified guanine to the end of the nascent RNA. Another protein (a phosphatase) removes one of the phosphate groups, and a third (a methyl transferase) adds a methyl group to the guanine to form the capped mRNA.
This capped mRNA is exported from the nucleus using a nuclear pore complex into the cell’s cytosol in eukaryotes. This step is facilitated by the aminoacyl-tRNA synthetase enzymes that attach the correct amino acid to each nucleotide sequence in the mRNA and the proteins that guide it through the pore complex. Finally, a particular mRNA-dependent splicing factor called EF-Tu discriminates among various aminoacyl-tRNA molecules. It directs only those with the correct coding sequence into the ribosome.
Translation
Your cells make proteins every second of the day, and each protein requires a chain of amino acids connected just the right way. To do so, mRNA must first be translated into a polypeptide chain by two types of RNA molecules: transfer RNA (tRNA), which reads the genetic information contained in mRNA’s sequence of nucleotide triplets (“codons”) and carries them to ribosomal rRNA, the central component of the ribosome protein-manufacturing machinery.
In translation, an mRNA sequence starts in one of three possible places on the ribosome, each identified by a codon (nucleotide triplet) that codes for a particular amino acid. During translation, the tRNA carrying the first codon binds to the ribosomal rRNA at the P site. As a result, a methionine molecule is added to the ribosome, and a fresh codon is exposed in another ribosomal rRNA slot, known as the A site.
The GTP-bound elongation factor (EF-Tu) escorts the aminoacyl tRNA to the A site, which matches its anticodon to the appropriate amino acid in a binding, GTP hydrolysis, and dissociation cycle. As this occurs, the tRNA-aminoacyl-tRNA complex triggers additional conformational changes in the ribosome, and the A site is ready to receive the next incoming amino acid.
Assembly
Once mRNA is transcribed, it must be assembled into the complex molecular setup, allowing protein production. During this step, the initiator tRNA (bearing methionine) binds to the 5′ cap of the mRNA and scans from the start of the coding sequence to the 3′ end, looking for the correct start codon. Once found, the large ribosomal subunit joins the complex to complete assembly. This complex is then ready for translation.
This process is facilitated by a feature in mRNA called a Shine-Dalgarno sequence, which marks the starting position of each coding sequence. This sequence ensures that the ribosome starts at the right place on the mRNA and does not get stuck on any one section, which would halt protein synthesis.
In the downstream process, the mRNA is purified to remove salts, cap analogs, NTPs, proteins, residual plasmid DNA, and dsRNA. This is achieved through a series of unit operations bracketed by two tangential flow filtrations and diafiltration (UFDF). This ensures the removal of unwanted contaminants while maintaining product purity and an acceptable chromatography loading condition.