Gene Expression In E. coli
The promoter in transcription will determine where transcription will begin. It will also dictate how efficient the transcription will be i.e. how much is made. In E. coli the mRNA is polycistronic as opposed to monocistronic in eukarya. This means that the mRNA can transcribe several proteins at once rather than every protein sequence having it's own transcription promoter and transcription terminator as in eukaryotic organisms. The beginning of the transcribed region of the mRNA is designated +1 and has consensus sequences upstream at -10 and -35 which determine the strength of the promoter involved in translation.
The perfect consensus sequence will deliver a very strong promoter, however, no consensus sequence in E. coli has this. The further away from the perfect sequence e.g. if a mutation occurs, the weaker the promoter. E. coli has 2 consensus sequences which are 16-19bp's apart. They are -10 (TATAAT) and -35 (TTGACA). 5- 8 base pair after the -10 sequence is start site(+1), it is TG/AT.
The Transcription Bubble and Elongation
The core RNA polymerase cannot bind directly to the promoter region without a sigma factor attached, creating a holoenzyme. Sigma factor 70 is the most common but there are others e.g. sigma factor 32 (involved in heat shock) and sigma factor 54. The transcription bubble moves along the dsDNA separating the strand as the holoenzyme is also a helicase. The core RNA polymerase is the subunit that undertakes elongation of the mRNA at a rate of ~20-30nts per second under normal physiological conditions.
N.b. The RNA polymerase has no proof reading function and the error rate is ~1 in 10,000. The RNA is only used to make proteins and then it is degraded i.e. it is not passed on to progeny.
Termination of Transcription
Transcription is terminated in one of two ways:
1) Factor Independant Termination - in the DNA there is a sequence of 4-10 A-T base pairs and a pallendromic sequence of G-C rich areas. This will cause the formation of a hairpin loop in the mRNA reducing the contact between the RNA and the DNA. Affinity is reduced and the RNA polymerase slows causing the dissociation of the RNA polymerase from the DNA.
2) Rho Dependant Termination - Rho factor is composed of 6 subunits and is a helicase. It 'unzips' RNA-DNA complexes or RNA-RNA complexes as it is a helicase and it is known as the weak termination factor.
Regulation of Transcription
Regulation of gene transcription is needed primarily to conserve the energy of the cell. Transcription can be regulated by either repression or activation. Repression of transcription is caused by a repressor protein bound to the promoter region of the operon and when removed will cause transcription of the gene - this is a negative acting factor. The Lac repressor protein in the lac operon is an example of this. Activation is needed because of a weak promoter; the activator enhances the initiation of transcription by the promoter - this is a positive acting factor.
Translation is process of creating the protein through the formation of peptide bonds between amino acids. Translation requires tRNA molecules (adaptors) which 'carry' a specific amino acid. These complexes are called aminoacyl (charged) tRNAs. This joining is catalysed by tRNA synthetases which are energy dependant enzymes. tRNA molecules contain modified bases that are either methylated or dimethylated as an alternative to the normal A, C, U and G. This makes some parts of the tRNA hydrophobic (Berg, J., Tymoczko, J and Stryer, L.: 860). These adapted molecules are inosine, dihydrouridine, pseudouridine and ribothymidine.
The Wobble Hypothesis
Base pairing occurs between the first two bases of the anticodon on the tRNA and the codon at the 5' end. However, inosine is a modified base and can pair with A, C or U at the 3' end.
The Composition of Prokaryotic Ribosomes
Ribosomes are large ribonucleoproteins that move along the mRNA and align successive aminoacyl tRNAs. The RNA is rRNA. Ribosomes consist of a larger 50S subunit and a smaller 30S subunit which are involved in the initiation of translation.
Initiation of Translation
Initiation requires initiation factors and an initiator tRNA which carries a methionine amino acid. The 30S subunit requires IF2 in order to bind to the ribosome binding site (RBS), also known as the Shine-Dalgarno sequence which causes the release of IF3. The 50S subunit can then 'lock on' to the mRNA which causes the release of IF1 and IF2, the remaining initiator factors GTP is hydrolysed and the initiator tRNA (charged) binds to the first codon which is at the P site (peptidyl site).
Elongation of the Protein
Charged tRNAs carry amino acids to a vacant acceptor site; a peptide bond forms between the two amino acids in the P site and the A site respectively. This is catalysed by peptidyl transferase, a ribozyme that is a component of the 23S subunit.
Termination of the Protein
Release factors are needed to interact with the STOP codons in order to terminate the sequence. The release factors are specific to the type of STOP codon present. RF1 interacts with UAA and UAG and RF2 interacts with UAA and UGA. RF3 aids the other two release factors in their function. The uncharged tRNA is removed and the ribosome dissociates from the mRNA.