Lecture 12 (Linneweber) Flashcards
RNA export, ribosomes and translation
What are Ribozymes?
What are Ribozymes?
- Ribozymes are ribonucleic acid based enzymes (non-proteinaceous catalysts).
- Ribozymes are mostly auto-nucleolytic, they catalyse the hydrolysis of the RNA backbone in a sequence and/or structure specific manner
- artifical activities can/have been generated by SELEX strategies
Small Ribozymes
Examples & Reaction Mechanism
Examples
- Hammerhead, Hairpin, Hepatitis delta, glmS riboswitch, Twister, Pistol, Hatchet
Reaction Mechanism: Nucleolytic cleavage by Transesterification
- A small ribozyme catalyzes the cleavage of a phosphodiester bond.
- A basic group abstracts a proton from the 2′ hydroxyl group (-OH), activating it as a nucleophile.
- The 2′ hydroxyl performs a nucleophilic attack on the phosphorus atom in the phosphodiester bond.
- The Attack is supported by base, but requires a certain structure (in one line), for this sugar has to be tilted, this can only occur in ssRNA
- This forms a transient pentavalent phosphorus intermediate.
- The bond cleaves, generating a 5′ product with a cyclic 2′,3′ phosphate and a 3′ product with a free hydroxyl group.
- The ribozyme’s structure stabilizes the transition state and positions the reactants for efficient catalysis.
Common structure features for
small Ribozymes
Common structure features for small Ribozymes
- small ribozymes form junctions of 2-4 helices
- active RNA fragment about 100 nt
- stacking of bases in the junction
- some bases remain free; those contribute usually to activity
- Function: generate unit-length RNA genomes from
rolling circle replication products
The hammerhead ribozyme
The hammerhead ribozyme
- The hammerhead ribozyme was discovered in 1986 in plant satellite viruses and viroids.
- It functions in cleaving replication concatemers, which are long RNA strands produced during rolling-circle replication.
- The ribozyme has a secondary structure consisting of 3 helices (I, II, III) radiating from a central core, where the catalytic activity occurs.
- Primarily uses RNA folding for orientation to align reactive groups.
- Employs nucleobases (G12 and G8) as acid/base catalysts for cleavage
- Magnesium ions (Mg²⁺) bond in the calytic pocket are required for folding into its active structure, but not for catalysis itself
- Domain 2 mediates the coaxial alignment of helices II and III
- Domain 1 forms the active centre
- Cleavage occurs at a specific phosphodiester bond, producing RNA fragments essential for viral replication.
- Red: Highly conserved bases, Blue: Stem (sequences don’t matter)
Viroids
Viroids
- smallest known infectious agents, consisting of circular, single-stranded RNA molecules (240–400 nucleotides long).
- They lack a protein coat (naked RNA) and depend on host enzymes for replication.
- Replication occurs via rolling-circle replication in the nucleus or chloroplasts of host plants.
- Some viroids encode ribozymes (e.g., hammerhead ribozyme) to cleave replication intermediates.
- They cause plant diseases, leading to growth defects or stunted development (e.g., potato spindle tuber viroid).
- Unlike viruses, viroids do not encode any proteins and rely entirely on host machinery.
- Viroids are unique to plants and do not infect humans.
Viroid Life
Cycle
Viroid Life Cycle
- Viroid enters the cell and migrates to the chloroplast to evade the cytosolic RNA interference defense system.
- NEP (nuclear-encoded RNA polymerase) produces viroid RNA transcripts through rolling-circle replication.
- Hammerhead ribozymes facilitate self-cleavage of long RNA concatemers into unit-length RNA.
- The cleaved RNA strands undergo ligation to form circular RNA genomes.
- Circular RNA genomes are used for further replication and continue the cycle.
- Viroid-derived small RNAs (siRNAs) can be generated by DCL (Dicer-like proteins), contributing to RNA silencing pathways in the host.
Hairpin Ribozyme of Satellite RNA
Viruses
Hairpin Ribozyme of Satellite RNA Viruses
- The hairpin ribozyme is a small RNA enzyme found in satellite RNA viruses that cleaves RNA during the replication cycle.
- Loop-loop interactions align the scissile phosphate and the 2’-OH of the adjacent ribose for cleavage.
- needs divalent metals for the formation of the catalytic core, too
Mechanism
- A38 (acid) donates a proton to stabilize the leaving group.
- G8 (base) deprotonates the 2′-OH, activating it for nucleophilic attack on the adjacent phosphate.
- Forms a pentavalent transition state and cleaves the RNA, producing a 2′,3′ cyclic phosphate and a 5′-OH group.
- No metal ions are required; catalysis is RNA-driven.
- Generates unit-length RNA genomes during rolling-circle replication through self-cleavage.
- Commonly associated with plant viruses and requires co-infection with a helper virus for propagation.
Overview of Ribozyme Catalytic Strategies
Overview of Ribozyme Catalytic Strategies
Metal ion-assisted catalysis
- Metal ions (e.g., Mg²⁺) stabilize negative charges on the RNA backbone and assist in catalysis by positioning reactive groups or acting as Lewis acids.
RNA folding for orientation:
- Precise folding of the ribozyme aligns reactive groups, such as the 2′-OH and phosphate, to facilitate efficient catalysis.
Nucleobases as acids and bases:
- Certain nucleobases (e.g., G, A) act as proton donors or acceptors, driving the acid-base chemistry needed for phosphodiester bond cleavage.
Large Ribozymes
RNase P
RNase P
- RNase P is present in all three domains of life (Archaea, Eubacteria, and Eukaryota).
- It processes tRNA precursors by cleaving the 5′ leader sequence, essential for generating mature tRNAs.
- In vitro, its RNA component alone is catalytically active in some Archaea and Eubacteria.
- In E. coli, it consists of a 377-nucleotide RNA and a 119-amino acid protein.
- The catalytic core of RNase P is formed by RNA domains supporting each other through folding interactions.
- This folding makes the RNA take on a globular shape, similar to how proteins form a compact, functional core.
- RNase P activity relies on structural recognition of tRNA templates, particularly the conserved features of tRNA molecules.
The Ribosome
General
The Ribosome
- is a ribozyme, composed primarily of rRNA (two-thirds), with proteins providing structural support (not for catalytic function).
- It performs two main catalytic activities:
- Peptidyl-transferase for forming peptide bonds.
- Peptidyl-tRNA hydrolysis during translation termination, requiring release factors.
- Catalysis occurs in the large subunit (23S rRNA), where the active site contains no protein atoms.
- The small subunit (16S rRNA) is responsible for decoding mRNA.
- The ribosome orients amino-acylated tRNAs but does not chemically participate in the reaction.
The Ribosome
Mechanism
Mechanism
- The CCA sequence at the 3′ end of tRNAs interacts with specific regions of the 23S rRNA, ensuring proper positioning and forming the catalytic core.
- The ribosome aligns substrates at the A-site and P-site precisely, enabling efficient catalysis.
- The α-amino group of the A-site substrate aligns with residues in the 23S rRNA, facilitating peptide bond formation via proton shuttling.
- Catalysis is limited by substrate binding (entry of charged tRNAs), not by the reaction speed itself:
- Binding of aminoacyl-tRNA to the A-site occurs at ~10/sec.
- Peptide bond formation can occur at ~300/sec.
- Catalytic acceleration (10⁷-fold) is much lower than in protein enzymes (10²³-fold), but maybe RNA-catalysis survived because with the low binding rates, speed of catalysis did never matter in evolution
Structure of eukaryotic & prokaryotic Ribosomes
Structure of eukaryotic & prokaryotic Ribosomes
Initiation of Translation in
Eukaryotes
Initiation of Translation in Eukaryotes
- Eukaryotic mRNAs are monocistronic, meaning each mRNA encodes a single protein.
- the initiator tRNA–methionine complex (Met–tRNAi) binds to the small ribosomal subunit with the help of eukaryotic initiation factors (eIFs).
- Unlike other tRNAs, the initiator tRNA binds directly to the P site.
- The small ribosomal subunit attaches to the 5′ end of the mRNA, recognized by its 5′ cap, which is bound by eIF4E and eIF4G –> resulting in a circular mRNA.
- The ribosome scans the mRNA 5′ to 3′, searching for the first AUG start codon, assisted by ATP-powered helicases.
- When the start codon is found, initiation factors dissociate, and the large ribosomal subunit joins, completing the ribosome.
- Translation begins with the initiator tRNA at the P site and the A site vacant.
- Leaky scanning can occur if the start site differs from the Kozak sequence (5′-ACCAUGG-3′), allowing alternative AUGs to be used, producing proteins with different N-termini.
Initiation of Translation in
Prokaryotes
Initiation of Translation in Prokaryotes
- Bacterial mRNAs are polycistronic, encoding multiple proteins from the same mRNA molecule.
- Translation initiation uses the Shine–Dalgarno sequence, located a few nucleotides upstream of the start codon (AUG), has a consensus sequence 5′-AGGAGGU-3′.
- This sequence base pairs with the 16S rRNA of the small ribosomal subunit, positioning the start codon in the ribosome.
- Translation initiation factors help align the mRNA and assemble the large ribosomal subunit to form the complete ribosome.
- Bacterial ribosomes can initiate translation at internal start codons as long as they are preceded by a Shine–Dalgarno sequence.
Ribosome profiling (Ribo-seq)
Ribosome profiling
- maps ribosome positions on mRNAs, identifying actively translated mRNAs.
Process
- total RNA from a cell line or tissue is exposed to ribonucleases under conditions where only those RNA sequences covered by ribosomes are spared
- The protected RNAs are released from ribosomes, converted to DNA, and the nucleotide sequence of each is determined
- When these sequences are mapped on the genome, the positions of ribosomes across each mRNA species can be ascertained
Purpose
- Determines ribosome occupancy and position of all mRNAs
- shows that some mRNAs are abundant but not translated until an external signal is received.
- Identifies translated reading frames
- clarifies the translated space of a genome
