Lecture 19: Molecular Mechanism of Translation and Translational Control of Gene Expression

Molecular Mechanism of Translation and Translational Control of Gene Expression

Learning Objectives

  • Identify the locations of transcription and translation in prokaryotic and eukaryotic cells.

  • Describe the role of the ribosome during translation.

  • Identify the differences between mRNA, tRNA, and rRNA.

  • Evaluate translational and post-translational methods of gene expression control in relation to transcriptional methods.

Transcription & Translation: Location

  • Transcription Location in Cells:

    • Prokaryotes:

    • Occurs in the cytoplasm due to the absence of a nucleus.

    • DNA is located in the nucleoid region, and ribosomes are present freely within the cytoplasm.

    • Eukaryotes:

    • Occurs in the nucleus where DNA is enclosed.

    • Ribosomes are present in the cytoplasm and on the endoplasmic reticulum, where they participate in translation.

Transcription & Translation: Time

  • Simultaneous vs. Sequential Activity:

    • In prokaryotes, transcription and translation can occur nearly simultaneously, meaning RNA can be translated even as it is being synthesized.

    • In eukaryotes, transcription and translation can never occur simultaneously; transcription must first occur in the nucleus before translation takes place in the cytoplasm.

    • True or False Question:

    • In eukaryotes, transcription and translation can occur simultaneously.

      • Answer: False

Ribosome Function in Translation

  • Structure of Ribosomes:

    • Large Subunit: Responsible for forming peptide bonds between amino acids (the active site).

    • Small Subunit: Holds mRNA during translation.

  • Key Roles:

    • Large and small subunits join during translation, assisting in the assembly of amino acids into polypeptide chains.

Translation: tRNA

  • Transfer RNA (tRNA):

    • A specific RNA molecule that plays a critical role during translation.

    • Structure:

    • One end binds to an amino acid.

    • The other end has an anticodon, a triplet of ribonucleotides that pairs with the codon in mRNA.

    • Aminoacyl tRNA: Refers to tRNA when it is linked to its corresponding amino acid.

Translation: mRNA Binding Process

  1. mRNA Binding:

    • mRNA binds to the small subunit of the ribosome.

    • A region in mRNA known as the ribosome binding site pairs with a complementary sequence on the ribosome (small subunit).

  2. Anticodon Binding:

    • The anticodon of an aminoacyl tRNA binds to the start codon in the mRNA, facilitating the correct alignment for translation.

Initiation of Translation

  • The first aminoacyl tRNA that binds to the start codon carries the amino acid Methionine (Met), which initiates polypeptide chain synthesis.

    • The corresponding anticodon on this tRNA is AUG.

    • As the translation begins, the large ribosomal subunit assembles onto the small subunit, and the tRNA is positioned at the P site where peptide bond formation can occur.

Translation Process Steps

  1. The large subunit assembles onto the small subunit, positioning the initial aminoacyl tRNA at the P site.

  2. A new aminoacyl tRNA occupies the A site, and its anticodon pairs with the mRNA codon.

  3. A peptide bond forms between the amino acids at the A and P sites.

  4. tRNA molecules shift position as follows:

    • The tRNA in the P site moves to the E site (exit).

    • The tRNA in the A site moves to the P site.

Stop Codon Mechanism

  • The translation process continues until the ribosome encounters a stop codon.

  • A protein release factor recognizes the stop codon and fills the A site, which causes:

    • Release of the assembled polypeptide chain and tRNAs.

    • Separation of the ribosomal subunits, thus terminating the translation process.

What is a Ribosome?

  • Ribosome Composition:

    • Comprises about equal amounts of protein and RNA.

    • The active site where peptide bonds form is composed entirely of RNA, highlighting that ribosomes are examples of ribonucleoproteins that catalyze chemical reactions.\n - Question: What is a ribosome?

      • Answer Options:

        • a) Nucleic acid

        • b) Protein

        • c) Carbohydrate

      • Correct Answer: Nucleic acid.

Comparison: mRNA vs tRNA vs rRNA

  • Types of RNA:

    • rRNA (ribosomal RNA): The RNA that forms the structural and functional core of the ribosome.

    • tRNA (transfer RNA): The RNA molecule responsible for transporting amino acids to the ribosome for protein synthesis.

    • mRNA (messenger RNA): The product of transcription that carries genetic information from DNA to the ribosome.

  • Comparison Statements:

    1. A brain cell and a liver cell would have the same sequence of: mRNA.

    2. Translated into a protein: mRNA.

    3. Required for the process of translation: mRNA, tRNA, and rRNA.

    4. Transports amino acids: tRNA.

    5. Catalyzes the peptide bond between amino acids: rRNA.

Gene Expression Control

  • Cells regulate which genes are expressed to respond efficiently to environmental changes and maintain a unique identity, exemplified through differential gene expression.

  • All cells contain identical DNA, but variations in gene expression cause differences in cell types (e.g., skin cell vs. stomach cell).

Methods of Gene Expression Control

  • Transcriptional Control Methods:

    • Prokaryotes: Operons

    • Eukaryotes: Multilevel control including:

    • Level 1: Chromatin remodeling (via methylation/acetylation)

    • Level 2: Transcription factors and regulatory sequences

    • Level 3: mRNA alternative splicing

    • Translational Control: Additional gene expression controls post-transcription.

Translational Control: RNA Interference

  • RNA Interference:

    • A process through which mature mRNA is prevented from being translated, using single-stranded RNA (commonly microRNA) associated with RISC (RNA-induced silencing complex).

    • The steps involved in RNAi include:

    1. miRNA binds to RISC.

    2. The miRNA-RISC complex binds to a complementary sequence on a mature mRNA.

    3. RISC either inhibits or destroys the mature mRNA, preventing its translation.

RNA Interference in the Lab

  • RNA interference (RNAi) has been utilized to better understand gene function:

    • For instance, introducing a strand of RNA complementary to a target mRNA will:

    • a) Prevent the target gene from being expressed.

    • b) Over-express the target gene.

    • Correct Response: a) - The target gene will not be expressed.

Post-Translational Modifications

  • Proteins undergo various modifications post-synthesis to become functional:

    • Folding: Can occur spontaneously or be assisted by molecular chaperones (located in the Rough Endoplasmic Reticulum - RER).

    • Chemical Modifications: Additions of carbohydrates or other molecules usually occur in the RER.

    • Amino Acid Removal: Certain amino acids may be removed from the primary structure during processing, occurring in the Golgi Apparatus (GA) or cytoplasm.

Post-Translational Control: Folding, Cutting and Destroying

  • Incorrectly folded amino acid chains can lead to dysfunction; proteins may:

    • Fold incorrectly.

    • Be chopped (cleaved) into smaller fragments.

    • Have chemical groups improperly added or missing to prevent their functionality, thus controlling gene expression.

Post-Translational Control: Protein Inactivation

  • Proteins can be turned off or deactivated after they have been made; mechanisms of inactivation include:

    • Inhibitory molecules such as enzyme regulators or phosphate groups attach, inhibiting protein activity.

Gene Expression Control: Speed vs Efficiency

  1. Evaluation of control mechanisms based on energy efficiency and responsiveness includes questions on which is most effective:

    • a) Transcriptional control

    • b) Translational control

    • c) Post-translational control

  2. Scenario: Due to a lack of glucose, a cell is using lipid metabolism. Upon glucose availability:

    • Fastest response could be:

    • a) Inactivate proteins associated with lipid metabolism (most efficient response).

    • b) Prevent transcription of proteins associated with lipid metabolism.

Gene Expression Control: Putting it All Together

  1. In individuals with Myotonic Dystrophy, the MD1 gene’s protein often fails to stay functional despite successful transcription and translation. Likely control mechanisms include:

    • a) Different transcription factors

    • b) Alternative splicing

    • c) RNA interference

    • d) Folding, cutting, or destroying

    • e) Inactivating

  2. The gene IM2 is expressed in all white blood cells. Notably, mature mRNA lengths differ in monocytes (1,677 ribonucleotides) vs. lymphocytes (1,201 ribonucleotides), indicating:

    • Likely mechanisms of gene expression control involves:

    • a) Different transcription factors

    • b) Alternative splicing

    • c) RNA interference

    • d) Folding, cutting & destroying

    • e) Protein inactivation.