lecture #10 lecture

1. Overview of Splicing and Translation

1.1 RNA Splicing

  • Definition: RNA splicing is the process of removing introns (non-coding sequences) from pre-mRNA and joining together the remaining exons (coding sequences) to form mature mRNA.

  • Process:

    • Transcription creates a precursor mRNA (pre-mRNA) that contains both introns and exons.

    • Exon-Intron Structure: Pre-mRNA structure shown as: Exon 1-Intron 1-Exon 2-Intron 2-Exon 3...

    • Splicing involves breaking the junctions at exon-intron boundaries and connecting exons directly, resulting in a continuous mRNA.

1.2 Heterogeneous Nuclear RNA (hnRNA)

  • Definition: hnRNA represents the primary transcript of nuclear genes synthesized by RNA polymerase II.

    • Characterized by wide size distribution and low stability.

  • hnRNP: Complex of hnRNA with proteins; pre-mRNAs remain in the nucleus until fully processed.

1.3 mRNA Capping

  • The 5' end of eukaryotic mRNA is modified by adding a methylated guanine cap.

    • Function of the 5' Cap: Recognized by proteins that aid in mRNA stability, splicing, export, and translation.

1.4 Nuclear Splice Junctions

  • Short sequences at exon-intron boundaries critical for splicing.

    • Splice Sites: 5′ splice site includes 'GU' and 3′ splice site includes 'AG' (known as the GU-AG rule).

  • Recognition of Splice Sites: All 5′ and 3′ splice sites are functionally equivalent and splicing occurs at accurate junctions.

1.5 Mechanism of Splicing

  • Lariat Formation: Introns are removed by splicing at the branch site, forming a lariat structure.

  • Transesterification: A chemical reaction involved in forming bond transfers during splice processes.

1.6 Role of snRNA (small nuclear RNA)

  • Function: snRNAs are essential for splicing, forming a spliceosome with proteins (snRNPs) like U1, U2, U5, U4, and U6.

  • Spliceosome Activity: Forms a large complex that executes splicing.

1.7 Alternative Splicing

  • Definition: Alternation of exons includes/excludes specific sequences yielding diverse mRNA and protein products.

  • Benefit: Increases structural diversity of proteins and impacts gene expression.

1.8 Regulation of Splicing

  • Involves enhancers and silencers which affect splice site usage.

  • Proteins that bind to RNA can either promote or suppress the selection of splice sites, influenced by transcription rates and developmental cues.

2. Overview of Translation

2.1 Translation Process

  • Comprises three stages: Initiation, Elongation, and Termination.

  • Ribosomes facilitate translation, utilizing mRNA and tRNAs to synthesize polypeptides.

2.2 Ribosome Structure

  • Consists of two subunits (70S in bacteria, 80S in eukaryotes) containing rRNA and ribosomal proteins.

    • Key rRNA Components: 23S, 16S, 28S, etc. with significant contributions to ribosome function.

2.3 Stages of Translation

2.3.1 Initiation

  • Ribosomal subunits bind at the ribosome-binding site on mRNA, aligning with the start codon (AUG).

  • In eukaryotes, small subunit scans for the initiation codon using features like the Kozak sequence.

2.3.2 Elongation

  • Aminoacyl-tRNA enters the ribosome and the polypeptide transfer occurs from peptidyl-tRNA to growing chain.

  • Translocation moves ribosome along mRNA, shifting tRNA through A, P, and E sites.

2.3.3 Termination

  • Translation ends when a stop codon (UAA, UAG, UGA) is encountered.

  • Release factors (RF1, RF2) interact with the ribosome to release the completed polypeptide.

2.4 Accuracy Control Mechanisms

  • Various mechanisms ensure fidelity during translation, helping to maintain correct amino acid sequences.

2.5 Initiation Factors in Eukaryotes

  • Initiation involves multiple factors to aid in assembly and progression through the stages, ensuring accurate translation commencement.