Eukaryotic vs Prokaryotic

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RNA Synthesis and Transcription Process

  • RNA is synthesized in the 5' to 3' direction using a DNA template.

  • Transcription Steps:

    1. Initiation: Binding of RNA polymerase to the promoter region of the DNA.
    2. Elongation: RNA chain is synthesized as ribonucleotides (rNTPs) are added.
    3. Termination: Completed RNA molecule is released.

  • Building Blocks: rNTPs serve as the building blocks for RNA synthesis.

  • No Primer Needed: Unlike DNA replication, RNA synthesis does not require a primer.

  • Promoter Function: Promoters are DNA sequences that control where transcription begins, consisting of consensus sequences that are recognized by the transcription machinery.

Central Concept: Translation

  • Translation Definition: The process of synthesizing a protein (polypeptide) from mRNA.
  • Location: Occurs in the cytoplasm and is performed by ribosomes (which comprise rRNA and proteins).
  • Genetic Code Utilization: The genetic code is read in sets of three nucleotides (codons), each coding for an amino acid.
Genetic Code Details

  • Codon Structure: Each codon consists of three nucleotides read in the 5' to 3' direction.

  • Degeneracy: The genetic code is degenerate, meaning one amino acid can be coded by multiple codons (e.g., Leucine can be encoded by CUU or CUA).

  • Codon Count: There are 4 nucleotides (A, U, G, C), resulting in 64 codons that map to 20 amino acids and several stop signals.

    • Start Codon: AUG (codes for Methionine)
    • Stop Codons: UAA, UAG, UGA

  • Reading Frames: Each strand of RNA has three potential reading frames, but only one is correct for producing a functional protein, beginning at AUG and concluding at a stop codon.

tRNA (Transfer RNA)
  • Structure: tRNA is a single-stranded RNA molecule that possesses an anticodon and a site for amino acid attachment at the 3' end.
  • Aminoacyl-tRNA Synthetase: This enzyme attaches the appropriate amino acid to its corresponding tRNA.
  • Charged tRNA: The tRNA-amino acid complex is referred to as charged tRNA.
Codon-Anticodon Pairing
  • Pairing Mechanism: The anticodon on tRNA pairs with the corresponding mRNA codon.
  • Wobble Position: The third base in codon-anticodon pairing allows for some flexibility, contributing to codon redundancy.
Ribosome Structure
  • Composition: The ribosome is made up of rRNA and proteins, containing the following sites:
    • A Site (Aminoacyl): Point of entry for charged tRNA.
    • P Site (Peptidyl): Holds the growing peptide chain.
    • E Site (Exit): Location where uncharged tRNA exits the ribosome.

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Initiation of Transcription

  • Holoenzyme Formation: Initiation involves a holoenzyme composed of a core enzyme (RNA polymerase) and a sigma factor, which binds to the promoter and ensures the correct positioning.
  • Transcription Bubble: Upon binding, a transcription bubble (12-14 bp) forms that allows access to the DNA template.
Elongation Phase
  • Synthesis Rate: RNA polymerase synthesizes RNA in the 5' to 3' direction at approximately 40 nucleotides per second.
  • Less Accuracy: RNA polymerase is generally less accurate than DNA polymerase, resulting in a higher rate of errors.
  • DNA-RNA Hybrid: Inside the polymerase, the DNA-RNA hybrid is about 8-9 bp long.
Termination of Transcription
  1. Rho Independent Termination (Intrinsic): A hairpin loop forms in the RNA, creating physical stress and weakening the U-A bonds to promote dissociation from DNA without protein factors.
  2. Rho Dependent Termination (Extrinsic): This process requires the Rho protein, which binds to the rut site on RNA and unwinds the DNA-RNA hybrid, enabling RNA release.
Note on mRNA Processing
  • There is no RNA processing in prokaryotes. mRNA produced immediately enters translation.
  • Both transcription and translation occur in the cytoplasm simultaneously, with generally fewer types of non-coding RNAs compared to eukaryotes. No splicing is necessary, indicating that genes are continuous.

Initiation of Translation

  • Shine-Dalgarno Sequence: Ribosome binding site upstream of the start codon (AUG) that facilitates proper ribosome alignment for translation.
  • Components of Initiation Complex: Includes the 30S ribosomal subunit, fMet-tRNA^fMet, mRNA, and initiation factors (IF-1, IF-2, IF-3) along with GTP.
  • Formation of Ribosome: Following the binding of the small subunit, the 50S subunit joins to create the complete 70S ribosome. A stronger Shine-Dalgarno match results in a more effective translation start.

Simultaneous Transcription & Translation

  • In prokaryotes, mRNA is translated immediately after synthesis due to the lack of a nuclear membrane.
  • Polyribosomes: Multiple ribosomes can simultaneously translate the same mRNA.
  • The ribosomes are 70S: consisting of a large 50S subunit and a small 30S subunit.
Translation Elongation
  • EF-Tu Function: Delivers aminoacyl-tRNA to the A site.
  • EF-Ts Role: Regenerates EF-Tu.
  • EF-G Action: Facilitates ribosome translocation in the 5’ to 3’ direction.
Translation Termination
  • Release Factors: RF1 recognizes stop codons UAA and UAG; RF2 recognizes UGA and UAA; RF3, in conjunction with GTP, helps dissociate the ribosome.

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Eukaryotic Transcription vs Prokaryotic Transcription

  • No Sigma Factor: In eukaryotes, initiation requires transcription factors (e.g., TFIID binds to the TATA box).
  • Enzyme Utilization: Transcription in eukaryotes is performed by RNA polymerase II.
  • Complex Termination: Eukaryotic termination mechanisms are more intricate, often involving cleavage and polyadenylation.
Eukaryotic Transcription Initiation
  • Basal Transcription Factors: Bind to the core promoter (e.g., TATA box) and include proteins like TFIID needed to recruit RNA polymerase II for minimal transcription levels.
  • Regulatory Transcription Factors: Bind to regulatory promoters/enhancers to enhance transcription levels.
  • Core Promoter: More complex than bacterial promoters and contains various conserved elements such as:
    • TATA box (~25 nt)
    • TFIBB recognition element
    • Initiator element
    • Downstream core promoter elements (positive/negative regulatory roles).
RNA Capping (5' CAP)
  • 5' Capping: A GTP molecule is added in reverse orientation to the first nucleotide by a capping enzyme linked to RNA polymerase II and methylated by methyl transferase.
  • Function of 5' Cap: Protects mRNA from degradation, aids in ribosome binding, and signals for nuclear export.
3' Poly-A Tail (Polyadenylation)
  • Addition of Adenines: 50-250 adenine nucleotides added to the 3' end in two steps:
    1. Cleavage downstream of the AAUAAA signal.
    2. Addition of adenines by poly-A polymerase.
  • Functions of Poly-A Tail: Provides stability, assists in ribosome positioning, and promotes nuclear export.
Termination via Polyadenylation
  • Cleavage yields a free 5' end, while Rat1, a 5' -to-3' exonuclease, degrades the remaining RNA, causing RNA polymerase II to detach and end transcription.
RNA Splicing
  • Post-Transcription Process: Involves the removal of introns (non-coding regions) and joining of exons (coding regions) to produce mature mRNA.
  • EXONS: Present in both genomic DNA and mature mRNA, they represent coding sequences.
  • INTRONS: Found in genomic DNA but not in mature mRNA, they interrupt coding sequences.
Spliceosome Complex
  • Structure: Composed of snRNPs (small nuclear ribonucleoproteins) and proteins that identify consensus sequences at the 5’ splice site, branch point, and 3' splice site to form a lariat loop for intron removal.

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Alternative Splicing

  • Definition: One pre-mRNA may undergo splicing in various ways to generate different mature mRNAs, leading to multiple protein isoforms with different structural and functional characteristics.

  • Mechanisms of Alternative Splicing:

    • Exon skipping
    • Mutually exclusive exons
    • Alternative 5' or 3' splice sites
    • Retained introns

  • Outcomes of Alternative Splicing: Increases protein diversity and is significant in cell-type-specific expression, development, and immune responses.

Non-coding RNAs (ncRNAs)

  • Functional Role: ncRNAs are not translated into proteins but serve important regulatory and structural roles.
  • Examples of ncRNAs:
    • rRNA: Structural component of ribosomes.
    • snRNPs: Function in splicing.
    • miRNA/siRNA/piRNA: Play roles in gene silencing.
    • incRNA: Involved in regulatory functions.
    • Telomerase RNA: Component of the telomerase enzyme.
  • Synthesis Location: ncRNAs are synthesized in the nucleus and are involved in transcriptional/post-transcriptional regulation.

Eukaryotic Translation Initiation

  • Absence of Shine-Dalgarno Sequence: Eukaryotes utilize the 5' cap and poly-A tail for ribosome recognition and stabilization.
  • Role of eIFs: Eukaryotic initiation factors (eIFs) assist in the binding of the 40S ribosomal subunit to mRNA and scan for AUG within a Kozak sequence (e.g., ACCAUGG).
  • Formation of 80S Ribosome: This ribosome comprises a 40S small subunit and a 60S large subunit.

Ribosome Composition

  • Composition Summary: Ribosomes are made up of ribosomal RNA (rRNA) and proteins, forming an 80S ribosome composed of a 60S large subunit and a 40S small subunit.
  • Assembly Location: Subunits are assembled in the nucleolus and function in the cytoplasm.

Post-Translational Modifications

  • Definition: Occur after translation to render proteins functional, which include:
    • Phosphorylation (addition of phosphate groups)
    • Protein folding and formation of disulfide bonds.
    • Joining with other polypeptides (e.g., hemoglobin subunits).
  • Modification Sites: These processes typically take place in the endoplasmic reticulum and Golgi apparatus.