Gene expression: Eukaryotes

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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.

 EUKARYOTIC GENE EXPRESSION

Unlike prokaryotes, eukaryotic cells have nucleus. This means that transcription and translation cannot occur at the same time. Transcription is carried out inside the nucleus of the cell.

Transcription

 a) Initiation
The initiation of transcription in eukaryotes requires the participation of the promoter region, transcription factors, and RNA polymerase.

There are several core promoter elements in eukaryotes. The most commonly studied is the TATA box. This is located 25-30 base pairs upstream from the transcription start site. The TATA box is where the transcription factors bind.

Transcription factors are composed of several proteins. The first transcription factor to bind to the promoter region is the TATA-binding protein (TBP), which is a subunit of  Transcription Factor II D (TFIID). Other transcription factors will then bind around the promoter region. These transcription factors are: TFIIA, TFIIB, TFIIE, TFIIF, and TFIIH. TFIIH is involved in separating the double-stranded DNA providing RNA polymerase access to the a single-stranded DNA template.

 There are three RNA polymerases in in eukaryotes. RNA polymerase I is used to produce ribosomal RNA (rRNA). RNA polymerase II synthesizes all protein-coding nuclear pre-mRNAs. RNA polymerase III transcribes structural rNAs such as 5s pre-rRNA, pre-tRNA, and small nuclear pre-RNAs. For transcription of mRNA, polymerase II binds to the transcription factors on the promoter region.


The assembly of the transcription factors, the promoter, and RNA polymerase forms the transcription pre-initiation complex (PIC). Once the PIC is complete, elongation is now ready to proceed.



b) Elongation
The process of transcription elongation in prokaryotes and eukaryotes are the same. However, eukaryotic transcription involves extra steps due to the extensively packaged DNA template.

 In eukaryotes, the DNA strand is wound twice around  eight charged histone proteins . For synthesis to occur, the transcription machinery need to move the histones out to loosen the strand allowing transcription to occur. The removal of histones is accomplished by the protein dimer FACT (facilitates chromatin transcription). FACT  partially disassembles the nucleosome upstream of the transcribing RNA Polymerase II. This is accomplished by removing two histones (H2A and H2B) out. FACT  reassembles the nucleosome behind the the RNA Polymerase II by returning the two histones back.

Once the nucleosome is partially disassembled, this allows RNA Polymerase II to form a "transcription bubble". RNA Pol II acts as it's own helicase, sliding clamp, single-stranded DNA binding protein among many other functions. RNA Pol II unwind the DNA ahead of it and rewind behind it. When the transcription start region has been unwound, the RNA Pol II is positioned at the +1 initiation nucleotide. At this point, the transcription initiation proteins are removed and elongation proceeds. RNA Polymerase uses the DNA strand below them as a template strand. During synthesis, G is paired with C, T is paired with A, and A is paired with U. Synthesis occurs in the 5' to 3' direction. 

c) Termination
Termination of transcription for protein encoding genes is facilitated by cleaving. The cleaving site occurs between an upstream AAUAAA sequence and a downstream GU-rich sequence separated by about 40-60 nucleotides. When these sequences are transcribed, a CPSF proteins binds to the AAUAAA seqeunce and the proetin CstF binds to the GU-rich sequence. The CPSF complex will cleave the pre-mRNA between the two proteins. The Poly(A) Polymerase (PAP), which is part of the complex will begin to add a Poly-A tail at the 3' end of the RNA strand.

 An exonuclease (Xrn2 protein) will attack the 5' end of the RNA strand still associated with the Polymerase. Xrn2 will continue digesting the non-released RNA strand until it reached the RNA polymerase.  This aids in displacing the RNA polymerase from the DNA strand terminating transcription.

 The addition of the poly-A tail at the 3' end and the cap at the 5' end produces the mature mRNA. It is now ready to be transported outside of the nucleus for translation.

Translation

 a) Initiation
Like in prokaryotes, eukaryotic translation initiation requires the formation of the initiation complex (mRNA template, small subunit of the ribosome (40S), initiation factors,  initiator tRNA) and the GTP as the energy source.
In eukaryotic translation initiation, the initiation complex recognizes the 7-methylguanosine cap at the 5' end of the mRNA. A cap-binding protein together with the initiation factors facilitates the binding of the ribosome to the 5' cap.  The Initiation complex then tracts along the mRNA in the 5' to 3' direction to search for the start codon AUG.

Once AUG is located, proteins and CBP dissociates. The larger subunit (60S) then binds to the complex positioning Met-tRNAi at the P-site. This completes the translation initiation phase.



 b) Elongation
Elongation proceeds with the new charged tRNA entering into the A-site. The ribosome moves along the the mRNA template in the direction of 5' to 3'. As the ribosome moves, the tRNA from the A-site moves to the P-site and the tRNA from the P-site moves to the E-site and is released. Polypeptide bonds form between the amino acids attached to the A-site tRNA and P-site tRNA. Energy used for polypeptide bond formation is derived from the hydrolysis of GTP to GDP. As the ribosome moves along the mRNA template, the amino acid chain continues to grow.

 c) Termination
Translation termination happens when a stop codon (UAG, UGA, UAA) enters the A-site. A release factor then binds to the stop codon. This facilitates the addition of a water molecule to the carboxyl end of the P-site amino acid. When this happens, the P-site amino acid detached from its tRNA. A newly formed protein is then released. The small and large ribosomal subunits dissociate from each other and the mRNA template.
Differences in Prokaryotic and Eukaryotic gene expression:

  1. ribosomes: 30S and 50S in prokaryotes, 40S and 60S in eukaryotes
  2. initiator tRNA: fMet in prokaryotes, Met in eukaryotes
  3. translation initiation complex binding site: Shine-Delgarno in prokaryotes, 7-methylguanosine cap in eukaryotes
  4. transcription and translation process: simultaneous in prokaryotes, not simultatneous in eukaryotes


 References:
[2]https://www.nature.com/scitable/topicpage/gene-expression-14121669/
[3]https://courses.lumenlearning.com/boundless-biology/chapter/eukaryotic-transcription/
[4]https://courses.lumenlearning.com/wm-biology1/chapter/reading-steps-of-translation/
[5]https://www.khanacademy.org/science/biology/gene-expression-central-dogma/translation-polypeptides/a/the-stages-of-translation

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