Gene expression: Prokaryotes

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Gene expression is  the molecular process by which DNA  is converted into a functional product called proteins[1]. The two key steps in the production of proteins is Transcription (DNA to RNA) and Translation (RNA to proteins)[1][2]. The processes are different in prokaryotes and eukaryotes.

PROKARYOTIC GENE EXPRESSION

Transcription

a) Initiation
In prokaryotes, like bacteria, the chromosome is a covalently-closed circle. For transcription to occur, the DNA double helix must partially unwind. The unwound region is called the transcription bubble. A holoenzyme (a fully functioning enzyme) and promoter is necessary for the initiation of transcription[3].

Prokaryotic RNA polymerase is the holoenzyme which assembles each time a gene is transcribed, and disassembles once transcription is complete. It is composed of 5 polypeptide subunits. These subunits are α, α, β, β', and σ[3].
  • α-subunits are used to assemble the polymerase on the DNA
  • β-subunit binds to the ribonucleoside triphosphate that will become part  of the nascent "recently-born" mRNA molecule
  • β'-subunit binds the DNA template strand
  • σ-subunit (specificity factor) ensures specific binding of RNA polymerase to the promoter region. This subunit ensures transcription on the correct site to produce the correct proteins later on.
The region where transcription begins is called the initiation site (+1 site). At the -10 and -35 regions upstream of the the initiation site are consensus sequences called promoters. The consensus sequence at the -10 region is the TATAAT. The consensus sequence at the -35 region is the TTGACA. It is in the -35 region where the σ-subunit (sigma factor) binds. When RNA polymerase has bound to the site, the initiation phase will start. First thing that happens is the unwinding of the double helix, forming a 'transcription bubble'. This is facilitated by the A-T-rich -10 region. Initially, a polymer of 10 nucleotides (abortive transcript) will be made and released. This signals the end of the initiation phase[3].


b) Elongation
Transcription elongation begins when the σ-subunit of the RNA polymerase is released. This allows the RNA polymerase to move along the DNA template. Synthesis of mRNA occurs in the 5' to 3' direction at the rate of approximately 40 nucleotides per second[3].

During elongation, the double helix is continuously unwound ahead of the core enzyme and rewound behind it. During synthesis, C is paired with G, T is paired with A, and A is paired with U.


c) Termination
Transcription is terminated when RNA polymerase dissociates, and the new mRNA is released. Termination is signaled either by a protein or an RNA.

The protein-based termination signal is controlled by the rho- protein. This termination is best known as the Rho-dependent termination. In this type of termination, the rho protein attaches to a recognition site (rut - rho utilization site) on the nascent mRNA and tracks along behind the RNA polymerase. The RNA polymerase will encounter a series of G nucleotides on the DNA template, causing it to stall. When the polymerase stalls, the rho protein collides with the polymerase. The rho protein unwinds the DNA-RNA hybrids in the transcription bubble. This releases the RNA strand. The rho protein and RNA polymerase then dissociates[3][4].












The RNA-based termination is called the rho-independent termination. This type of termination is controlled by specific sequences in the DNA template strand. Near the end of transcription, the polymerase will encounter a C-G-rich region. Because these nucleotides are complimentary to each other, it will fold back on itself forming a stable hairpin. This causes the polymerase to stall as soon as it begins to transcribe the A-T-rich region of the template.  The complimentary U-A strand in the nascent mRNA forms a weak interaction with the template DNA. This causes instability for the core enzyme which then dissociates releasing the new mRNA transcript[3].


Translation

Because the DNA of a prokaryote is not membrane-bound, translation in prokaryotes can happen while transcription is occurring[3].

a) Initiation
Translation begins with the formation of the initiation complex which is composed of:
  • mRNA template
  • Large(50S) and (30S) small ribosomal subunit
  • Initiation factors - assists in the proper assembly of the ribosome
  • Guanosine-triphosphate (GTP) - energy source
  • tRNA with N-formyl-methionine (fMet) - initiator tRNA
The first step of translation initiation is the binding of the initiator tRNA with the codon AUG (start codon). Upstream of the start codon, is the leader sequence, also known as the Shine-Delgarno sequence. This is also known as the ribosomal binding site AGGAGG. Complimentary base pairs found in the ribosome anchors the 30S ribosomal subunit followed by the 50S ribosomal subunit. This forms the intact ribosome. When the ribosomes are attached to the mRNA, the initiator tRNA is contained in the P site of the intact ribosome[5]. Each step in translation initiation requires energy. Energy is acquired through the hydrolysis of GTP to GDP + Pi.



b) Elongation
The intact ribosome forms three sites necessary during elongation. These are:

  • A (aminoacyl) site - binds incoming charged aminoacyl tRNAs.
  • P (peptidyl) site - binds charged tRNAs carrying amino acids that has formed peptide bonds with growing polypeptide chain.
  •  E (exit) site - releases dissociated tRNAs
Translation elongation proceeds through translocation events. In a translocation event, a charged tRNA enters the A site. Through conformational changes, the ribosome advances by three base pairs in the 3' direction. This moves the charged tRNA from the A site to the P site, and the tRNA from the P site will be moved to the E site. Another charged tRNA will then enter the now vacant A site.

The amino acids carried by these tRNAs will be attached to each other by forming peptide bonds. This is catalyzed by the enzyme peptidyl transferase. The tRNA at the E site is detached from it's amino acid, then expelled. Energy used to perform the process of elongation is through the hydrolysis of GTP[5].

Translocation events will continue until translation is ready to be terminated.



c) Termination
Termination happens when  a nonsense codon is encountered. The nonsense codons are as follows:
  • UAA
  • UAG
  • UGA
When the nonsense codon is in the A site, a release factor comes in contact. This triggers the release of the amino acid from the tRNA on the P site. The newly formed polypeptide chain is released. The small and large ribosomal subunits then dissociates from each other, and from the mRNA[5].
 













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