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Genes Expression - Module 2 Reading / Watching

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Course: An introduction to Gene Regulation and Gene Expression
Book: Genes Expression - Module 2 Reading / Watching
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Date: Wednesday, 4 June 2025, 10:41 AM

Table of contents

1. Gene expression

In this book you will see an overview of the topics of Module Two and reference links.

By the end of this book, you will be able to:

    • Discuss flow of information in biological systems 
    • Explain the process of Transcription and Translation  in Prokaryotes
    • Discuss about Eukaryotic Transcription and Translation
    • Analyze the use of inhibitors of Transcription and Translation in medical field

1.1. Central Dogma of Molecular Biology -Information flow in biological systems

The central dogma of molecular biology  explains the flow of genetic information within a biological system. It is often stated as “DNA makes RNA, and RNA makes protein” .

  •  DNA can be copied to DNA through a molecular process called DNA Replication. Instructions for making  proteins with the correct sequence of amino acids are encoded in DNA.
  •  This information in DNA can be copied into RNA  through the molecular process called  by which the information in the DNA of every cell is converted into small, portable RNA messages.
  •  Proteins can be synthesized using the information in mRNA as a template through the molecular process called  During translation, the RNA messages travel from DNA  in the nucleus to the ribosomes in the cytoplasm where they are ‘read’ to make specific proteins.

1.2. Prokaryotic Transcription

The process of  transcription occurs in the nucleus of the cell in eukaryotes  and the mRNA transcript must be transported to the cytoplasm for protein synthesis . In prokaryotes, which lack membrane-bound nuclei and other organelles, transcription occurs in the cytoplasm of the cell. Therefore, the processes of transcription and  translation,  can all occur simultaneously. This is referred to as  . The intracellular level of a bacterial protein can quickly be amplified by multiple transcription and translation events occurring concurrently on the same DNA template. Prokaryotic transcription often covers more than one gene and produces polycistronic mRNAs that specify more than one protein.
The Process of Prokaryotic Transcription

The first step in transcription is initiation, when the RNA pol binds to the DNA upstream (5′) of the gene at a specialized sequence called a promoter .

Prokaryotic Promoters
  • The DNA sequence onto which the proteins and enzymes involved in transcription bind to initiate the process is called a. Promoters usually exist upstream of the genes they regulate. The specific sequence of a promoter determines whether the corresponding gene is transcribed all of the time, some of the time, or hardly at all. The structure and function of a prokaryotic promoter is relatively simple 
  • The -10 consensus sequence or the Pribnow box  : In prokaryotes, most genes have a sequence called the Pribnow box, with the consensus sequence TATAAT positioned about ten base pairs away from the site . This serves as the location of transcription initiation. Not all Pribnow boxes have this exact nucleotide sequence; these nucleotides are simply the most common ones found at each site.
  • The -35 consensus sequence : Many genes also have the consensus sequence TTGCCA at -35  position, upstream of the start site
  • Upstream element  : an A-T rich region 40 to 60 nucleotides upstream that enhances the rate of transcription

The process of transcription starts with the binding of  the RNA pol “holoenzyme” binding to the template DNA and  unwinds  the DNA double helix in order to facilitate access to the gene. The sigma subunit conveys promoter specificity to RNA polymerase; that is, it instructs the  RNA polymerase where to bind. There are a number of different sigma subunits that bind to different promoters. These sigma subunits assist in turning genes on and off as conditions change.

The mRNA product is complementary to the template strand and is almost identical to the other DNA strand, called the non-template strand, with the exception that RNA contains a uracil (U) in place of the thymine (T) found in DNA. Like DNA polymerase, RNA polymerase adds new nucleotides onto the 3′-OH group of the previous nucleotide. This means that the growing mRNA strand is being synthesized in the 5′ to 3′ direction.

Elongation

Once transcription is initiated, the DNA double helix unwinds and RNA polymerase reads the template strand. Elongation phase begins with the release of the σ subunit from the polymerase. The dissociation of σ allows the core enzyme to proceed along the DNA template, synthesizing mRNA in the 5′ to 3′ direction .

Nucleotides  are added to the 3′ end of the growing chain . At a temperature of 370 C new nucleotides are added at an estimated rate of about 42-54 nucleotides per second in bacteria. As elongation proceeds, the DNA is continuously unwound ahead of the core enzyme and rewound behind it. The base pairing between DNA and RNA is not stable enough to maintain the stability of the mRNA synthesis components. Instead, the RNA polymerase acts as a stable linker between the DNA template and the nascent RNA strands to ensure that elongation is not interrupted prematurely.

Termination

Once a gene is transcribed, the prokaryotic polymerase needs to be instructed to dissociate from the DNA template and liberate the newly made mRNA. Depending on the gene being transcribed, there are two kinds of termination signals namely :

  1. Rho-dependent or Extrinsic termination : Termination is controlled by the rho protein.The rho protein tracks along behind the polymerase on the growing mRNA chain. Near the end of the gene, the polymerase encounters a run of G nucleotides on the DNA template and it stalls. As a result, the rho protein collides with the polymerase. Rho unwinds the DNA-RNA hybrid formed during transcription and releases the mRNA from the transcription bubble.
  2. Rho-independent or Intrinsic termination is controlled by  C–G nucleotide sequences in the DNA template strand at the end of the gene being transcribed. Due to the self complementary nature of the C-G nucleotides . The mRNA folds back on itself, and form a stable hairpin . This causes the polymerase to stall and  it begins to transcribe a region rich in A–T nucleotides. The complementary U–A region of the mRNA transcript forms only a weak interaction with the template DNA. This fact together  with the stalled polymerase, induces enough instability for the core enzyme to break away and liberate the new mRNA transcript.

Upon termination, the process of transcription is complete. By the transcription would end the prokaryotic transcript would  have already commenced the   synthesis of numerous copies of the encoded protein .


1.3. Eukaryotic Transcription

Initiation of Transcription by RNA Polymerase II
Initiation-Formation of the Transcription complex
  • The first step in formation of a transcription complex is the binding of a general transcription factors called TFIID to the TATA box.TFIID is itself composed of multiple subunits,  including the TATA binding protein . This binds specifically to the TATAA consensus sequence .and 10-12 other polypeptides, called TBP-associated factors (TAFs).
  • TBP then binds a second general transcription factor (TFIIB) forming a TBP-TFIIB complex at the along with TF II F at the promoter
  • Then RNA polymerase II binds
  • This is followed by the binding of TFIIE and TFIIH.
  • TFIIH is a multi subunit factor. It plays  two important roles. First, two subunits of TFIIH are helicases, which unwind DNA  around the initiation site. These subunits of TFIIH are also required for Nucleotide excision repair .Another subunit of TFIIH is a  Protein kinase . This phosphorylates repeated sequences present in the C-terminal domain of the largest subunit of RNA polymerase II. 
  • Once the complex is assembled, RNA polymerase can bind to its upstream sequence.
  • When bound along with the transcription factors, RNA polymerase is phosphorylated. This releases part of the protein from the DNA .  The transcription initiation complex  gets activated and places RNA polymerase in the correct orientation to begin transcription.
  • Then the double-stranded DNA in the transcription start region  is unwound . The RNA Polymerase II is then  positioned at the +1 initiation nucleotide and starts new RNA strand synthesis.

Elongation

  • RNA Polymerase  clears or “escapes” the promoter region and leaves most of the transcription initiation proteins behind.

  • RNA Polymerases travel along the template DNA strand in the 3′ to 5′ direction .The synthesis of new RNA strands takes place in the 5′ to 3′ direction. i.e.,  new nucleotides are added to the 3′ end of the growing RNA strand.

  • RNA Polymerases unwind the double stranded DNA ahead of them and rewinds the unwound DNA behind them.
  • RNA strand synthesis occurs in a transcription bubble of about 25 unwound DNA base pairs.
  • About 8 nucleotides of newly-synthesized RNA remain base paired to the template DNA. The rest of the RNA molecules falls off the template  and this  allows the DNA behind it to rewind.
Termination
  • The termination of transcription is different for the three different eukaryotic RNA polymerases.
  • RNA Polymerase I contain a specific sequence of base pairs -11 bp long in humans  :  18 bp in mice .  This sequence is recognized by a termination protein called TTF-1 (Transcription Termination Factor for RNA Polymerase I.).  This protein binds the DNA at its recognition sequence and blocks further transcription, causing the RNA Polymerase I to detach from the template DNA strand and  releases the newly-synthesized RNA.
  • RNA Polymerse II lack any specific signals or sequences that direct RNA Polymerase II to terminate at specific locations. The transcript is cleaved at an internal site before RNA Polymerase II finishes transcribing. The cleavage site occurs between an upstream AAUAAA sequence and a downstream GU-rich sequence separated by about 40-60 nucleotides in the emerging RNA.
  •  The upstream portion of the transcript is released .
  • The remainder of the transcript is digested by a 5′-exonuclease (called Xrn2 in humans) while it is still being transcribed by the RNA Polymerase II. When the 5′-exonulease “catches up” to RNA Polymerase II by digesting away all the overhanging RNA, it  disengage the polymerase from its DNA template strand, finally terminating transcription.
  • A protein called CPSF in humans binds the AAUAAA sequence and a protein called CstF in humans binds the GU-rich sequence. CPSF cleaves the nascent pre-mRNA at a site 10-30 nucleotides downstream from the AAUAAA site. The Poly(A) Polymerase enzyme  catalyze the addition of a 3′ poly-A tail on the pre-mRNA .
  • The RNAs transcribed by RNA Polymerase III have a short stretch of four to seven U’s at their 3′ end. This somehow triggers RNA Polymerase III to both release the nascent RNA and disengage from the template DNA strand.


1.4. Prokaryotic Translation

Initiation
  • During translation initiation, the ribosome recruits an mRNA and selects the start codon of the Open Reading Frame (ORF) 
  • m RNAS of prokaryotes have an extended 5′ untranslated region (5′UTR) and an (SD) located 8–10 nt upstream of the start codon (usually AUG).
  • Interactions between the SD sequence and the complementary anti-SD (aSD) sequence in 16S ribosomal RNA (rRNA) recruits the small 30 S ribosomal subunit and anchors  the 30S ribosomal subunit at the correct location on the mRNA template.
  • Initiation is promoted by initiation factors IF1, IF2, and IF3. IF1 enhances the activities of IF2 and IF3.IF2 is a GTPase that recruits the initiator fMet-tRNAfMet. IF3 interferes with subunit association. It also ensures the fidelity of fMet-tRNAfMet selection over the elongator aminoacyl-tRNAs (aa-tRNAs), and helps to discriminate against mRNAs with unfavorable translation initiation regions (TIRs)
  • The initiator tRNA then interacts with the start codon AUG (or rarely, GUG). This tRNA carries the amino acid methionine, which is formylated after its attachment to the tRNA and thus the  Charged t RNA , fMet-tRNAMetf    is formed .  This is mediated by the initiation factor IF-2.
  • The initiating fMet is usually removed after translation is complete.
  • After the formation of the initiation complex, the 30S ribosomal subunit is joined by the 50S subunit to form the 70S translation complex. 
    Elongation
  • Elongation includes repetitive cycles of decoding the m RNA , peptide bond formation, and translocation.
  • Elongation begins as soon as the second codon of the ORF becomes accessible for reading by the next amino acylated tRNAs 
  •  Elongation is facilitated by  translation factors EF-Tu, EF-G ,EF-Ts EF-P and EF-4.
  • The second  codon  in the P site is recognized by aminoacylated -tRNAs.  A ternary complex of  aa-tRANA ,EF-Tu and GTP is attached to the P site .This interaction triggers GTP hydrolysis by EF-Tu. After Pi release, EF-Tu rearranges into the guanosine diphosphate (GDP)-bound form and releases the aa-tRNA.
  • The amino group of the amino acid attached to the A-site tRNA  makes a peptide bond with the carboxyl group of the amino acid attached to the P-site tRNA. This reaction is catalyzed by peptidyl transferase, an RNA-based ribozyme that is integrated into the 50S ribosomal subunit. Now the A site carries a peptide comprising of two amino acids and leaves the uncharged t RNA in the p site .
  • During elongation the ribosomes move one codon along the mRNA in a process called Translocation. Translocation is promoted by EF-G at the cost of GTP hydrolysis .During each translocation event, a new  charged tRNAs enter at the A site, then shift to the P site, and then finally to the E site for removal. Ribosomal movements  are induced by conformational changes that advance the ribosome by three bases in the 3′ direction.
  • This process of elongation continues until the ribosome arrives at the stop codon.
  • Amazingly, the E. coli translation apparatus takes only 0.05 seconds to add each amino acid, meaning that a 200 amino-acid protein can be translated in just 10 seconds.

Termination
  • Termination occurs when the ribosome encounters a stop codon UAG/UAA and UGA/UAA, respectively in the mRNA.
  • These stop codons are recognized by the termination (or release) factors RF1 and RF2.
  • Another termination factor, RF3, facilitates turnover of RF1 and RF2 but is not required for peptidyl-tRNA hydrolysis.
  • The mechanism of termination encompasses  three steps:
    • recognition of the stop codon,
    • hydrolysis of the ester bond of the peptidyl-tRNA (these two steps are accomplished by RF1 or RF2) and
    • dissociation of RF1/RF2 with the help of RF3.
  • The releasing factors instruct peptidyl transferase to add a water molecule to the carboxyl end of the P-site amino acid. This reaction forces the P-site amino acid to detach from its tRNA, and releases the  newly made protein.
  • The small and large ribosomal subunits dissociate from the mRNA and from each other; they are recruited almost immediately into another translation initiation complex. After many ribosomes have completed translation, the mRNA is degraded so the nucleotides can be reused in another transcription reaction.



1.5. Eukaryotic Translation

Eukaryotic Translation-Initiation
The process of initiation can be grouped in to 4 main headings as
  • Ribosome dissociation in to 40 S and 60 S
  • Ternary complex  called pre initiation complex formation – initiator t RNA +GTP+ eIF2+40S ribosomal subunit
  • mRNA binding to preinitiation complex
  • 60S ribosomal subunit binding to the complex
The steps in the process of initiation
The steps can be summarized as :
  • GTP binds to eIF2- Binary complex
  • eIF2 composed of 3 subunits – α, β and γ.
  • Binary complex binding to initiator t RNA
  • Binding of 40S ribosomal subunit
  • Ternary complex – called 43S preinitiation complex.  This complex is stabilized by earlier  association of eIF3 and eIF1 to the 40S subunit
  • Eukaryotes does not require the Shine-Dalgarno sequence rather the eukaryotic initiation complex recognizes the 7-methylguanosine cap at the 5′ end of the mRNA.
  • Cap structure of mRNA is bound by eIFs prior to pre initiation complex formation. Cap binding accomplished by e IF -4F
    This factor is a complex of 3 proteins  namely  eIF-4E, A and G 
    eIF-4E    –    24 KDa protein, recognizes and binds cap structure
    eIF-4A   –    46 KDa protein, binds and hydrolyses ATP , exhibits RNA helicase activity, resolves RNA secondary structures.
    eIF- 4G    – helps in the binding of mRNA to 43S preinitiation complex
  • The cap-binding protein (CBP) and  IFs assist the movement of the ribosome to the 5′ cap. Once at the cap, the initiation complex moves  along the mRNA in the 5′ to 3′ direction, searching for the AUG start codon.
  • Many eukaryotic mRNAs are translated from the first AUG, but this is not always the case.
  • According to Kozak’s rules, the nucleotides around the AUG indicate whether it is the correct start codon.  state that the following consensus sequence must appear around the AUG of vertebrate genes: 5′-gccRccAUGG-3′. The R (for purine) indicates a site that can be either A or G, but cannot be C or U. Sequences closer to this consensus show higher efficiency of translation.
  • e IF-5 binds to preinitiation complex. This is followed by the binding of initiator t RNA – met –tRNA met   to the AUG codon of mRNA ( process helped by eIF-1)
  • This is followed by the binding of 60S subunit and results in the formation of 80S complex.
  • Association of 60S requires e IF-5.Energy for the binding dervied by GTP hydrolysis – bound to eIF-2.
Eukaryotic Translation-Elongation & Termination

The sequence of steps in the process of elongation are :
•Movement of ribosome to next codon
•Incoming aminoacyl tRNA brought to the ribosome by eEF-1α- GTP.
•GTP hydrolysed
•eEF-1α- GDP- complex dissociates
•GDP/ GTP exchange for additional translocations.( carried out by eEF-1 βγ).
•Peptide in ribosome P-site transferred to aminoacyl-tRNA in A site.
•Reaction  called Transpeptidation ; catalysed by peptidyl transferase.
•Peptidyl tRNA movement from A site to P site called –Translocation. Catalyzed by eEF-2: coupled to GTP hydrolysis
•In the process of translocation- Ribosome moved to next codon  of mRNA  resides in the A site.
•eEF-2 released. Cycle begins again.
Termination
•Requires releasing factors – eRFs
•Termination signal – UAG, UAA and UGA
•eRF binds to A site  along  with GTP
•This binding stimulates peptidyl transferase activity to transfer the peptidyl group to water
•Uncharged t RNA left in p site expelled  with GTP hydolysis
•80S complex dissociates