Comparison of Vector Cloning, PCR and Sanger's DNA Sequencing

Translation

Act I: Initiation

• The mRNA comes to the cytoplasm, with a starting codon AUG.
• A small ribosomal subunit binds to mRNA. The ribosome has two ribosomal units (small and large). 
• The first tRNA carrying a specific amino acid, met, at one end and having a specific nucleotide triplet, anticodon, UAC, at the other end comes to bind to the starting codon.
• The ribosome also has one site for binding of mRNA and three sites for the binding of tRNA (P site, A site, E site).

1. A site: new tRNA with next amino acid to be added to the chain.
2. P site: holds growing polypeptide chain.
3. E site: discharged tRNA which will return to cytoplasm and pick up designated amino acid.

• Initiation factors brings the large ribosomal subunit to mRNA, placing the tRNA in the P site.

Act II: Elongation

• Another tRNA carrying amino acid recognizes its corresponding codon at the A site.
• An RNA molecule catalyzes the formation of a peptide bond between the polypeptide in the P site with the new amino acid in the A site.
• The polypeptide chain is transferred to the tRNA at the A site.
• The ribosome moves the tRNA with the attached polypeptide from the A site to the P site. This process need energy provided by GTP.
• The first tRNA enters the E site, and as the third tRNA attaches to the A site, it exits the E site to the cytoplasm to pick another amino acid.

Act III: Termination

• When one of the three stop codons UAG, UAA, UGA reaches the A site, a release factor cut the bond between the polypeptide chain and its tRNA at the P site.
• Polypeptide, which is known as protein, is released.
• Translation complex disassembles.

Transcription

Transcription is the copying of a sequence of DNA to produce a complementary strand of RNA

Act I: Initiation

• Transcription factors recognize promoter regions (TATA box) on the template and bind to the promoters.
• This signals RNA polymerase II to bind to transcription factors. Together they form a transcription initiation complex.
• Polymerase II starts transcription.

Act II: Elongation

• RNA polymerase unwinds DNA double helix and adds RNA nucleotides to the 3' end of the growing strand. RNA, same as DNA, grows from 5' to 3'.
• When adding the complimentary nucleotides to the RNA transcript, every thymine is replaced by uracil.
• As RNA polymerase II moves forward, the double helix behind re-forms, and the newly transcribed RNA molecule, RNA transcript, peels away.
• The strand that RNA reads is called the template strand/ antisense strand.
• The strand that has exactly same sequence as RNA is called coding strand/ sense strand.
• A single gene can be transcribed by multiple polymerase simultaneously.

Act III: Termination

• Transcription stops when RNA polymerase transcribes as a terminator AAUAA.
• The pre-mRNA is cut, and RNA polymerase II is released form the DNA.
• In eukaryotic cells, before the mRNA enter the cytoplasm, enzymes work on modifying them.
• G-cap (modified guanine) is assed to the 5' end of pre-mRNA.
• PolyA-tail is added to the 3' end of pre-mRNA.

• Pre-mRNA combines with snRNPs and other proteins to form a spliceosome.
• The snRNA in the snRNPs base pair with nucleotides at the end of the noncoding segments on the pre-mRNA, introns.
• Introns are excised from the pre-mRNA, then the coding regions, exons, are spliced together in the spliceosome.

• The snRNA acts as a ribozyme, and the RNA acts as an enzyme.
• The spliceosome comes apart, releasing mRNA.