Structural Biochemistry/Bacterial Protein Synthesis

Overview of Bacterial Gene to protein
The DNA has two strands, a sense strand and a template strand. The sense strand has the same sequence as the mRNA that will be transcribed, except the T on the DNA will be replaced with U’s on the mRNA. RNA Polymerase will make a complementary mRNA transcript from the template strand of DNA.

Transcription

 * 1) Initiation: RNA polymerase will move along the DNA, looking for the -35 region and -10 region of the sigma-70 promoter in E.Coli. Once it finds the promoter, RNA polymerase will bind to the promoter, loosely at first then more tightly once DNA starts to unwind. RNA polymerase will then add a ribonucleoside triphosphate (rNTP), usually a purine. This rNTP will be complementary to the nucleotide on the +1 position of the DNA template.
 * 2) Termination: The transcription termination site is located downstream from the translation stop codon. In bacteria, there are two types of terminations possible:
 * Rho dependent:
 * A Rho factor will bind to the RNA in a region, called the transcription terminator pause site-- this is rich in guanine and cytosine and is after the part of the gene that codes for protein. Rho will then wrap the downstream RNA (the RNA between where Rho binds and the RNA polymerase) around itself and slowly pull itself to the RNA polymerase, which is now paused. When Rho comes into contact with the RNA polymerase, termination occurs and the mRNA transcript and RNA polymerase are released from the DNA template.
 * Rho independent-
 * A region of the mRNA transcript that is rich in guanine and cytosine forms a RNA stem loop that will hold onto the RNA polymerase and cause it to pause. During this pause, the poly-U and poly-A base pairs on the 3’ end of the mRNA is weak and therefore easy to melt. Transcription is stopped when the molecule is melted, and the mRNA transcript and RNA polymerase will be released.

Translation

 * 1) Initiation: For bacteria, initiation factors (IF) are involved in the initiation of translation. IF3 will bring mRNA and the 30S subunit of ribosome together. The ribosome binding site on the mRNA can then bind the complementary sequence on the 16S rRNA. IF1 will bind to the A site of the 30S ribosomal subunit and block that A site. IF2 that is attached to GTP can then bring the initiatior fMet-tRNA (N-formylmethionyl-tRNA) to the start codon on the P site of the 30S ribosomal subunit. With the attachment of the initiator tRNA, IF3 will be released and then the 50S subunit of the ribosome will be attached to the 30S. This leads to the hydrolysis of the GTP and therefore the release of the IF2 and IF1. The ribosome continue through translation.
 * 2) Termination: The ribosome will encounter a stop codon-- either UAA, UAG, or UGA, which appears in the A site of the ribosome. Instead of a tRNA binding, a protein release factor, either RF1 or RF2, will enter the A site of the ribosome. Peptidyltransferase will then cut the bond between the finished protein and the P site. Once the protein is released from the ribosome, RF3 will cause the protein release factor used to leave the ribosome. After, a ribosome recycling factor (RRF) and a bound EF-G will bind at the A site of the ribosome. GTP hydrolysis will take apart the 30S and 50S ribosomal subunit. IF3 will then bind to the 30S to remove any tRNA or mRNA left on the subunit. There is now a synthesized bacterial protein and ribosomal subunits that can help in further translations.