Genetics and Evolution: Molecular Basis of Inheritance - Initiation of Protein Synthesis

• Protein synthesis is a complex process that involves the translation of genetic information into functional proteins.
• In this lecture, we will focus on the initiation of protein synthesis and the role of various molecules and factors in this process.
• Understanding the initiation of protein synthesis is crucial as it is the first step in gene expression and plays a significant role in determining protein production.
• Let’s delve deeper into this topic and explore the molecular basis of initiation of protein synthesis.

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Slide 2: Central Dogma of Molecular Biology

• The central dogma of molecular biology describes the flow of genetic information from DNA to RNA to protein.
• According to this principle, DNA serves as the template for RNA synthesis, and RNA serves as the template for protein synthesis.
• Protein synthesis can be divided into three main stages: initiation, elongation, and termination.
• Initiation is the first stage, and it involves the assembly of ribosomes at the start codon of the messenger RNA (mRNA) molecule.

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Slide 3: Start Codon and AUG

• The start codon AUG, which codes for the amino acid methionine, marks the beginning of protein synthesis.
• AUG is recognized by the initiation complex, which includes the small ribosomal subunit, mRNA, and initiator tRNA.
• The initiator tRNA carries a special methionine called N-formylmethionine (fMet) in prokaryotes, and methionine in eukaryotes.
• AUG serves as a universal start codon in most organisms, but there are exceptions like mitochondria which use alternative start codons.

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Slide 4: Role of Initiator tRNA

• Initiator tRNA plays a crucial role in the initiation of protein synthesis.
• It binds to the start codon on mRNA and brings the first amino acid to the ribosome.
• Initiator tRNA in prokaryotes carries formylmethionine (fMet), while in eukaryotes, it carries methionine.
• The presence of initiator tRNA ensures accurate protein synthesis by specifying the correct reading frame for mRNA translation.

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Slide 5: Initiation Factors in Protein Synthesis

• Several initiation factors are involved in the initiation of protein synthesis in both prokaryotes and eukaryotes.
• In prokaryotes, three initiation factors (IF-1, IF-2, and IF-3) are required for the assembly of the initiation complex.
• In eukaryotes, initiation factors like eIF1, eIF2, eIF3, and eIF4E play crucial roles in binding the mRNA and assembling the ribosome.

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Slide 6: Shine-Dalgarno Sequence in Prokaryotes

• In prokaryotes, the Shine-Dalgarno sequence is a conserved sequence found in the 5’ untranslated region (UTR) of mRNA.
• It interacts with the ribosomal RNA (rRNA) of the small ribosomal subunit and facilitates the assembly of the initiation complex.
• The Shine-Dalgarno sequence helps in positioning the start codon in the ribosomal binding site, ensuring accurate initiation of protein synthesis.

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Slide 7: Kozak Sequence in Eukaryotes

• In eukaryotes, the Kozak sequence is a consensus sequence found around the start codon in mRNA.
• It plays a crucial role in the recognition of the start codon by the ribosome and ensures accurate initiation of protein synthesis.
• The Kozak sequence consists of the nucleotides surrounding the start codon (A/GCCRCCAUGG), with the most conserved element being the AUG start codon itself.

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Slide 8: 5’ Cap and Poly(A) Tail in mRNA

• In eukaryotes, mRNA molecules undergo post-transcriptional modifications before they can be used for protein synthesis.
• These modifications include the addition of a 5’ cap and a poly(A) tail.
• The 5’ cap protects the mRNA from degradation and helps in its recognition and binding by the ribosome during initiation.
• The poly(A) tail enhances mRNA stability and facilitates the binding of initiation factors during the initiation of protein synthesis.

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Slide 9: Assembly of the Initiation Complex

• The assembly of the initiation complex involves the binding of initiation factors and the small ribosomal subunit to the mRNA.
• In prokaryotes, the small ribosomal subunit binds to the Shine-Dalgarno sequence, while in eukaryotes, it binds to the 5’ cap structure.
• The initiator tRNA binds to the start codon on mRNA, bringing with it the first amino acid (fMet in prokaryotes, methionine in eukaryotes).
• This assembly marks the initiation of protein synthesis and primes the ribosome for the elongation phase.

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Slide 10: Summary

• Protein synthesis involves the translation of genetic information into functional proteins.
• The initiation of protein synthesis is the first step and involves the assembly of ribosomes at the start codon on mRNA.
• Start codons like AUG (AUG for methionine or N-formylmethionine) mark the beginning of protein synthesis.
• Initiator tRNA, initiation factors, Shine-Dalgarno sequence (in prokaryotes), and Kozak sequence (in eukaryotes) play crucial roles in initiation.
• Post-transcriptional modifications like the addition of a 5’ cap and a poly(A) tail also contribute to the initiation of protein synthesis.

Slide 11: Initiation Complex in Prokaryotes

• In prokaryotes, the assembly of the initiation complex begins with the binding of the small ribosomal subunit to the Shine-Dalgarno sequence on mRNA.
• The Shine-Dalgarno sequence is complementary to a specific region of the 16S rRNA in the small ribosomal subunit.
• Initiation factors (IF-1, IF-2, and IF-3) promote the binding of the ribosomal subunit and the initiator tRNA to the mRNA.
• IF-1 helps in the dissociation of ribosomal subunits, IF-2 delivers initiator tRNA, and IF-3 prevents premature joining of the subunits.
• The initiation complex formation is completed with the binding of the large ribosomal subunit, which forms the functional ribosome.

Slide 12: Initiation Complex in Eukaryotes

• In eukaryotes, initiation of protein synthesis is a more complex process than in prokaryotes.
• The initiation complex assembles at the 5’ cap structure of mRNA, with the help of initiation factors like eIF1, eIF2, and eIF3.
• eIF2 plays a critical role in delivering the initiator tRNA to the ribosomal subunit, while eIF3 helps in the binding of the small ribosomal subunit.
• Additional initiation factors, such as eIF4E and eIF4G, are involved in recruiting ribosomes to the mRNA and promoting translation initiation.
• The assembly of the initiation complex in eukaryotes is more regulated and allows for finer control of gene expression.

Slide 13: Regulation of Translation Initiation

• Translation initiation is a highly regulated process that allows cells to control protein production.
• Various mechanisms are involved in regulating translation initiation, including the availability of initiation factors and the presence of regulatory elements in mRNA.
• Some regulatory elements in mRNA can inhibit or enhance translation initiation by interacting with specific proteins or RNA molecules.
• Small regulatory RNAs, such as microRNAs (miRNAs) and small interfering RNAs (siRNAs), can bind to mRNA and prevent its translation.
• The regulation of translation initiation is crucial for the proper functioning of cells and for responding to different cellular conditions.

Slide 14: Metabolic Control of Protein Synthesis

• Protein synthesis can be regulated based on the metabolic needs of the cell.
• During times of nutrient abundance, cells can stimulate protein synthesis to support growth and proliferation.
• Conversely, during nutrient scarcity or stress conditions, protein synthesis can be downregulated to conserve energy and resources.
• Signaling pathways, such as the mTOR (mammalian target of rapamycin) pathway, play a key role in coordinating protein synthesis in response to nutrient availability.
• Dysregulation of metabolic control of protein synthesis can contribute to diseases such as cancer and metabolic disorders.

Slide 15: Examples of Initiation Complex Regulation

• Examples of regulation of the initiation complex include:
  • Phosphorylation of initiation factors: Phosphorylation can modulate the activity of initiation factors, affecting their ability to bind to mRNA or ribosomes.
  • RNA secondary structures: Some mRNAs contain structures that hinder the binding of ribosomes, inhibiting translation initiation.
  • miRNA-mediated regulation: miRNAs can bind to specific mRNA sequences and prevent translation initiation by either promoting mRNA degradation or inhibiting ribosome binding.
  • Internal ribosome entry sites (IRES): Some viral RNAs use IRES sequences that allow translation initiation independent of the 5’ cap, allowing for efficient protein synthesis during viral infection.

Slide 16: Cap-Dependent vs. Cap-Independent Initiation

• Cap-dependent initiation is the most common form of translation initiation in eukaryotes.
  • It involves the recognition of the 5’ cap structure by initiation factors, particularly eIF4E.
  • The cap-binding proteins assist in assembling the ribosome at the start codon by scanning along the mRNA.
  • It allows for efficient and regulated translation initiation.
• Cap-independent initiation is less common and is mainly observed in viral RNAs or specific cellular mRNAs.
  • It utilizes internal ribosome entry sites (IRES) or alternative mechanisms to bypass the need for a 5’ cap.
  • Cap-independent initiation enables the efficient translation of specific mRNAs under certain circumstances.

Slide 17: Summary

• The initiation of protein synthesis is the first step in gene expression, involving the assembly of ribosomes at the start codon.
• Initiator tRNA, initiation factors, Shine-Dalgarno sequence (in prokaryotes), and Kozak sequence (in eukaryotes) play crucial roles in initiation.
• Post-transcriptional modifications like the addition of a 5’ cap and a poly(A) tail are important for mRNA recognition during initiation.
• The initiation complex in prokaryotes and eukaryotes differs in terms of the binding sites and the involved factors.
• Translation initiation is regulated by various mechanisms, including the metabolic state of the cell and the presence of regulatory elements in mRNA.
• Cap-dependent initiation is the primary mode of initiation, while cap-independent initiation occurs in specific cases.

Slide 18: Practice Questions

1. What is the role of the initiator tRNA in protein synthesis?
2. Name three initiation factors involved in the assembly of the initiation complex in prokaryotes.
3. How does the Shine-Dalgarno sequence contribute to the initiation of protein synthesis?
4. What is the function of the 5’ cap and the poly(A) tail in mRNA?
5. Describe one example of translational regulation in response to metabolic conditions.

Slide 19: Practice Questions (Continued)

6. Explain the difference between cap-dependent and cap-independent initiation.
7. How do miRNAs regulate translation initiation?
8. Which organelles use alternative start codons instead of AUG?
9. How does phosphorylation of initiation factors affect translation initiation?
10. What is the function of eIF2 in the initiation of protein synthesis in eukaryotes?

Slide 20: Further Reading

• Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K., Walter, P. (2014). Molecular Biology of the Cell (6th ed.). Garland Science.
• Lodish, H., Berk, A., Zipursky, S. L., Matsudaira, P., Baltimore, D., Darnell, J. E. (2000). Molecular Cell Biology (4th ed.). W. H. Freeman and Company.
• Kozak, M. (1987). An analysis of mammalian mRNA sequences: implications for those expressed in brain. Nucleic Acids Research, 15(18), 8125-8148.
• Jackson, R. J., Hellen, C. U. T., Pestova, T. V. (2010). The mechanism of eukaryotic translation initiation and principles of its regulation. Nature Reviews Molecular Cell Biology, 11(2), 113-127.
  </li> </ul> <h1 id="slide-21-factors-affecting-translation-initiation">Slide 21: Factors Affecting Translation Initiation</h1> <ul> <li>Translation initiation can be influenced by various factors, including: <ul> <li>Availability of initiation factors: The presence or absence of specific initiation factors can modulate translation initiation efficiency.</li> <li>Secondary structure of mRNA: Highly structured regions in the mRNA can impede ribosome binding and affect translation initiation.</li> <li>Regulatory elements in 5’ or 3’ UTR: Sequences within the untranslated regions of mRNA can regulate translation initiation by interacting with specific proteins or RNA molecules.</li> <li>Codon usage: The frequency of specific codons in mRNA can influence translation initiation rates.</li> <li>RNA modifications: Chemical modifications of RNA bases can affect the efficiency of translation initiation.</li> </ul> </li> </ul> <hr> <h1 id="slide-22-upstream-open-reading-frames-uorfs">Slide 22: Upstream Open Reading Frames (uORFs)</h1> <ul> <li>Upstream Open Reading Frames (uORFs) are short coding sequences present in the 5’ UTR of mRNA.</li> <li>uORFs can influence translation initiation by modulating the availability of initiation factors and ribosomes for the main coding sequence.</li> <li>Depending on the sequence and structure of the uORF, translation initiation can either be enhanced or repressed for the main coding sequence.</li> <li>This mechanism provides an additional layer of regulation to control gene expression at the translational level.</li> <li>Examples of genes regulated by uORFs include genes involved in stress responses and developmental processes.</li> </ul> <hr> <h1 id="slide-23-role-of-translation-initiation-in-cancer">Slide 23: Role of Translation Initiation in Cancer</h1> <ul> <li>Dysregulation of translation initiation can contribute to cancer development and progression.</li> <li>Oncogene activation or loss of tumor suppressor function can lead to aberrant translation initiation.</li> <li>Overactivation of translation initiation can promote uncontrolled cell growth, proliferation, and survival.</li> <li>Mutations in initiation factors or regulatory elements can dysregulate translation initiation in cancer cells.</li> <li>Targeting translation initiation pathways is an emerging therapeutic approach for treating cancer.</li> </ul> <hr> <h1 id="slide-24-cap-independent-initiation-examples">Slide 24: Cap-Independent Initiation Examples</h1> <ul> <li>Cap-independent initiation mechanisms are observed in specific cases, such as: <ul> <li>Internal Ribosome Entry Sites (IRES): Some viral RNAs and few cellular mRNAs contain structured IRES elements that allow direct ribosome binding, bypassing the need for a 5’ cap.</li> <li>Ribosome shunting: This mechanism involves ribosomes bypassing structured regions of mRNA and directly scanning to the main coding sequence.</li> <li>Direct interaction with initiation factors: Certain mRNAs can directly interact with initiation factors to recruit ribosomes for translation initiation.</li> <li>Alternative translation initiation codons: Some mRNAs can use non-AUG start codons for translation initiation under specific conditions.</li> </ul> </li> </ul> <hr> <h1 id="slide-25-translation-initiation-vs-elongation">Slide 25: Translation Initiation vs. Elongation</h1> <ul> <li>Translation initiation and elongation are two distinct stages of protein synthesis.</li> <li>Initiation is the formation of the ribosomal machinery at the start codon and bringing the first amino acid.</li> <li>Elongation refers to the sequential addition of amino acids to the growing polypeptide chain.</li> <li>Elongation involves the movement of ribosomes along the mRNA, decoding codons, and catalyzing peptide bond formation.</li> <li>The elongation phase continues until a termination codon is reached.</li> </ul> <hr> <h1 id="slide-26-termination-of-protein-synthesis">Slide 26: Termination of Protein Synthesis</h1> <ul> <li>Termination is the final stage of protein synthesis, where the ribosome recognizes the termination codon (stop codon).</li> <li>Termination codons (UAA, UAG, UGA) do not code for any amino acid but signal the end of translation.</li> <li>Release factors (proteins) bind to the termination codon and cause the release of the completed polypeptide chain from the ribosome.</li> <li>The ribosome dissociates into its two subunits, and the newly synthesized protein is released into the cellular milieu.</li> </ul> <hr> <h1 id="slide-27-post-translational-modifications">Slide 27: Post-Translational Modifications</h1> <ul> <li>Post-translational modifications (PTMs) are chemical modifications that occur on proteins after translation.</li> <li>PTMs determine the protein’s structure, function, localization, and stability.</li> <li>Examples of PTMs include phosphorylation, acetylation, methylation, ubiquitination, and glycosylation.</li> <li>PTMs can affect protein activity, protein-protein interactions, and protein stability.</li> <li>PTMs are crucial for many cellular processes, including cell signaling, gene expression, and protein degradation.</li> </ul> <hr> <h1 id="slide-28-importance-of-protein-synthesis">Slide 28: Importance of Protein Synthesis</h1> <ul> <li>Protein synthesis is essential for maintaining cellular function and integrity.</li> <li>Proteins are involved in a wide range of biological processes, such as: <ul> <li>Enzymatic catalysis: Proteins act as catalysts for biochemical reactions.</li> <li>Structural support: Proteins provide structural support to cells and tissues.</li> <li>Cell signaling: Proteins mediate signaling pathways and convey information within cells and between cells.</li> <li>Transport and storage: Proteins transport molecules across membranes and store essential molecules like oxygen and iron.</li> <li>Immune response: Proteins play a vital role in the immune response, including antibody production.</li> <li>Gene expression regulation: Proteins regulate gene expression by binding to DNA or RNA molecules.</li> </ul> </li> </ul> <hr> <h1 id="slide-29-protein-synthesis-disorders">Slide 29: Protein Synthesis Disorders</h1> <ul> <li>Aberrations in protein synthesis can lead to various diseases and disorders, including: <ul> <li>Inherited genetic disorders: Mutations in genes encoding translation initiation factors or ribosomal components can disrupt protein synthesis, leading to disorders like Diamond-Blackfan anemia and Shwachman-Diamond syndrome.</li> <li>Neurodegenerative diseases: Protein misfolding and aggregation can occur due to errors in translation, leading to diseases like Alzheimer’s, Parkinson’s, and Huntington’s.</li> <li>Cancer: Dysregulated translation initiation can contribute to tumor development and progression by promoting rapid cell growth and survival.</li> <li>Antibiotic resistance: Bacterial resistance to antibiotics can arise from mutations in proteins involved in translation, leading to decreased antibiotic efficacy.</li> </ul> </li> </ul> <hr> <h1 id="slide-30-summary">Slide 30: Summary</h1> <ul> <li>Translation initiation is the first step in protein synthesis and involves the assembly of ribosomes at the start codon.</li> <li>Factors influencing translation initiation include the availability of initiation factors, mRNA secondary structure, regulatory elements in mRNA, codon usage, and RNA modifications.</li> <li>Upstream Open Reading Frames (uORFs) in the 5’ UTR can regulate translation initiation efficiency.</li> <li>Dysregulation of translation initiation can contribute to</li> </ul> </div> </div> <script src="/reveal/dist/reveal.js"></script> <script src="/reveal/plugin/markdown/markdown.js"></script> <script src="/reveal/plugin/highlight/highlight.js"></script> <script src="/reveal/plugin/math/math.js"></script> <script src="/reveal/plugin/anything/plugin.js"></script> <script src="/reveal/plugin/notes/notes.js"></script> <script 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