in Modification Eukaryotes: RNA processing
mRNA processing (pp 111-115; 493-505) 1. 5' Capping 2. Poly(A) adenylation 3. splicing
Post-transcriptional
RNA Processing
1. 5' Cap 2. 3' Poly(A) tail addition 3. Splicing
Splicing
RNA Processing: 5' Cap
Eukaryotic mRNAs have a 5' cap formed by a 5'-5' linkage of a 7-methylguanylate (GTP) residue. Catalyzed by a dimeric capping enzyme, which associates with the phosphorylated CTD of RNA polymerase II. -Capping is specific for Pol II Since Pol I or III does not contain a CTD, the capping enzyme does not associate with either of these RNA polymerases 5' Cap functions: transport out of the nucleus initiation of translation protect from exonucleases
RNA Processing: Polyadenylation
G/U
Poly(A) polymerase (PAP) adds A residues to the 3' end 200-250 Adenosine residues can be added to make the complete Poly(A) tail Poly(A) tail functions to protect the mRNA from premature degradation by 3'5' exonuclease
RNA Processing: Splicing
RNA Processing: Splicing
Four conserved sequences have been found to be necessary to remove the introns: - 5' splice site at the exon/intron junction (GU) - Branch point (Adenosine): usually 25-50 bases from the 3' splice site - Pyrimidine-rich region upstream of the 3'splice site -3' splice site at the intron/exon junction (AG) Generally, the central region of the intron is unnecessary for splicing to occur.
Splicing: 2 Transesterification Rxns
In each transesterification reaction, one phosphodiester bond is exchanged for another. In the first reaction, the ester bond between the 5' phosphorous of the intron and and the 3' oxygen of exon 1 is exchanged for an ester bond with the 2' oxygen of the the branch-site A residue. In the second reaction, the ester bond between the 5' phosphorous of exon 2 and the 3' oxygen of the intron is exchanged for an ester bond with the 3' oxygen of exon 1, releasing the intron as a lariat structure and joining the 2 exons.
RNA Processing: Splicing
The splicing reaction is catalyzed by enzymes composed of a complex of small nuclear RNAs (snRNAs) and proteins, referred to as small nuclear ribonucleoprotein particles or snRNPs (pronounced snurps). The RNA components of snRNPs are abundant, Uridine-rich snRNAs and are referred to as U1, U2, U4, U5 and U6. They interact together to form the spliceosome. Each snRNA is associated with 6-10 proteins
snRNA Base-Pair w/Pre-mRNA & w/ One Another
The 5' region of U1 snRNA base pairs with the 5' splice site of premRNA. U2 base pairs to regions flanking the the "branch point" A -The branch point A itself, which is not base paired to U2 snRNA, "bulges out," allowing its 2' OH to participate in the first transesterification reaction.
RNA Processing: Spliceosome formation
Assembly of the spliceosome begins with the base-pairing of the snRNAs of U1 and U2 to the pre-mRNA. U4/U6 form a complex then associates with U5....This U4/U6/U5 complex then associates with the previously formed U1/U2/pre-mRNA complex to yield a spliceosome. Extensive rearrangement in the pairing of snRNAs and the pre-mRNA lead to release of U1 and U4. This forms a catalytically active spliceosome
RNA Processing: Spliceosome formation
Remember: U2 and branch point base pairing results in looping out of A residue
RNA Processing: Spliceosome formation
The catalytically active rearranged spliceosome mediates the first transesterification reaction that forms the 2',5'-phospodiester. Following another rearrangement of the snRNPs, the second transesterification reaction ligates the two exons in a standard 3',5' phoshodiester bond. The intron is released as a lariat structure associated with the snRNPs. This complex rapidly dissociates, and the individual snRNPs can participate in a new cycle of splicing. The lariat is rapidly degraded
lariat
Transcription and RNA processing are coordinated
Remember: The carboxyl-terminal domain (CTD) of RNA pol II is composed of multiple repeats of a seven-nucleotide sequence.
The long length of of the CTD allows multiple proteins to associate simultaneously with a single RNA polymerase II molecule. - i.e. Dimeric capping enzyme
Transcription and RNA processing are coordinated
In addition, RNA splicing and polyadenylation factors associate with the phosphorylated CTD. - The close association between RNA pol II and the RNA processing machinery enhances the rate and specificity of RNA processing.
Transcription and processing of the ovalbumin gene
DNA RNA
24% original transcript
Regulation of Pre-mRNA Processing
RNA Processing: Alternative splicing
Many eukaryotic genes can be sliced into different mature mRNAs, a phenomenon called alternative splicing. Different tissues express different forms of the same gene due to alternative splicing. Example: Fibronectin Gene: -Fibroblasts produce one type of the extracellular fibronectin protein that contain the EIIIB and EIIIA exons (encode for protein domains that allow the protein to interact with the cell surface). -Hepatocytes produce another type of fibronectin protein that lack these domains (EIIIB and A spliced out)...as a result the protein circulates in the blood. Both fibronectin isoforms are encoded by the same transcription unit, which is spliced differently in the two cell types to yield two different mRNAs. Can this occur in the same cell type?
RNA Processing: Alternative splicing
Tropomyosins are a family of highly conserved actin-binding proteins. They not only bind to actin, but also help to regulate myosin's interaction with actin's thin filaments. Proteins encoded from genes undergoing alternative splicing are very similar except in key regions that might affect ligand binding, location and activity.
How do the snRNPs know which exons to splice together? There are regulatory proteins that can recognize splicing enhancer sequences that favors the splicing of one exon over another.
need to be eliminated before the mRNA can be translated?
Main rationale used to explain the existence of introns is that it has allowed evolution to proceed at an increased pace. Exons frequently encode various domains of a protein which can be combined by DNA rearrangements to generate new proteins relatively quickly (exon shuffling)
What is the advantage of having introns that
Another level to regulate gene express. -Alternative splicing allows a variety of related proteins to be synthesized from a single gene
Prokaryotic Vs Eukaryotic Transcription
Both processes go from 5' to 3' Transcription initiates at a promoter Both utilize RNA polymerases Regulation of transcription initiation is the most common mechanism for control Both involve other transcription activators and repressor proteins that bind to specific DNA sequences and influence transcription rate
Prokaryotic Vs Eukaryotic Transcription
Eukaryotes have 3 different RNA polymerases RNA polymerases have more subunits; more complex Prokaryotic promoters are recognized by a subunit of the polymerase. In eukaryotes, the core promoter often contains a TATA box that is recognized by TBP. TBP recruits the RNA polymerase For prokaryotes, "promoter" refers specifically to the RNA polymerase- binding site. In eukaryotes, this refers to all of the protein recognition sites In prokaryotes genes are regulated by RNA polymerase plus 1 or 2 transcription factors. In eukaryotes, it is regulated by many transcription factors. In Eukaryotes, the pre-mRNA needs to be processed into a mature mRNA
Eukaryotic Translation
Initiation Elongation Termination
Ch.4: pp 123-131s Ch12: pp 515, 523 Ch16: pp 657-661
In eukaryotes, transcription and translation are separated in space and time
Eukaryotic Translation
Genetic code: identical tRNAs: same; charged by 20 different aminoacyl-tRNA synthetase (ARS) Ribosomes: larger subunits but identical functions More factors are involved in eukaryotic translation With the exception of translation initiation, the other steps are similar to prokaryotic translation.
Composition of Ribosomes
Ribosomes are large (103 kDa) RNA-protein complexes composed of two subunits. More than 80% of cellular RNA is ribosomal RNA The large subunit (60S) is composed of 3 rRNAs and 50 proteins The small subunit (40S) is composed of 1 rRNA and 33 Ribosomes
proteins
Eukaryotic Ribosomes are found in two places: Membrane attached Are bound to the rough endoplasmic reticulum (ER) Proteins that are in the secretory pathway are synthesized by these ribosomes Membrane unattached Are free in the cytosol Proteins in the nonsecretory pathway are synthesized by these ribosomes
Ribosomes Cytoskeleton Centriole Lysosome Flagellum Not in most plant cells
Plasma membrane
Mitochondrion
Nucleus
Rough endoplasmic reticulum (ER) Golgi apparatus
Smooth endoplasmic reticulum (ER)
Eukaryotic Ribosomes
Eukaryotic Ribosomes
"Free" ribosomes: Electron micrograph of "free" ribosomes in the cytosol Not attached to ER membrane Synthesized proteins likely released into cytosol where they will remain, or will be transported to the nucleus, or incorporated into proper organelle
Where does translation take place?
Eukaryotic Ribosomes
Translation: Protein Synthesized by Ribosomes
Growing polypeptide chain
Ribosome
tRNA mRNA
tRNA4 leaving tRNA7 arriving
Fig. 4-26
General Overview of Translation: Occurs in Three steps
1. Initiation - finding the start codon and assembling the ribosomal subunits 2. Elongation - reading the mRNA sequence and polymerizing the addition of corresponding amino acids to growing polypeptide chain 3. Termination - recognition of the stop codon and release of the new polypeptide
The ribosome has three important sites
3 Sites of Ribosome: A site P site E site
What is the first amino acid?
Eukaryotic Translation: Initiation
A special tRNA is used to carry the methionine to the start codon methionyl-tRNAimeth Initiator tRNA tRNAi Proteins are acetylated at the amino end, which blocks the amino end of the peptide Can bind to the P site Regular methionyl-tRNAMet is used for the elongation of the polypeptide chain Can only bind to the A site, not the P site The same aminoacyl-tRNA synthetase (MetRS) charges both tRNAs with methionine.
Eukaryotic Translation: Initiation
How does a ribosome know where to start translation on an mRNA? The small subunit and initiation factors recognize the 5'cap of the mRNA.
mRNA
5'
----//----ACCAUGG----------- 3'
Cap
Kozak sequence
mRNA contains a translation initiation sequence called the Kozak sequence -It is a 7 nucleotide sequence that includes the AUG start codon. -The first A, AUG, and last G are most critical determinants of the Kozak sequence. -Is usually located 100 nt from the 5'cap
Translation Factors
Role
Initiation
Prokaryotes Eukaryotes
IF-1, IF-2, IF-3 eIF-1, eIF-2, eIF-2B eIF-3, eIF-4A, eIF-4B eIF-4C, eIF-4F, eIF-5 eIF-6
Elongation Termination
EF-Tu, EF-Ts, EF-G eEF-1, eEF-1, eEF-2
RF-1, RF-2, RF-3
eRF 1 and 3
Eukaryotic Translation: Initiation
Eukaryotic initiation factors (eIFs), eIF3 and eIF6 bind to the small and large ribosomal subunits, respectively to prevent them from binding to each other without mRNA Ternary complex is formed: eIF2-GTP + met-tRNAimet eIF1A, ternary complex, and eIF3-40S subunit form the preinitiation complex. Cells can regulate protein synthesis by phosphorylating a serine residue on the eIF2 bound to GDP. -The phosphorylated complex is unable to exchange the bound GDP for GTP and cannot bind Met-tRNAiMet, thus inhibiting protein synthesis
Eukaryotic Translation: Initiation
As the mature mRNA is transported from the nucleus into the cytoplasm, initiation factors (IF4s) for protein synthesis binds to the 5'cap
Eukaryotic Translation: Initiation
4G 4A 4E 4B
AUG
(AAA)n
5'cap
Preinitiation complex binds to the mRNA-eIF4E complex to form the initiation complex through an interaction of the eIF4G subunit and eIF3. The initiation complex then scans along the mRNA to look for the Kozak sequence containing the first AUG
Eukaryotic Translation: Initiation
As the initiation complex scan the mRNA, the helicase eIF4A uses ATP to unwind 2o RNA structures Scanning stops as the met-tRNAimet anticodon recognizes the AUG codon (Kozack sequence). eIF2-GTP hydrolyzes to eIF2-GDP, an irreversible step that prevents further scanning. The eIFs dissociates eIF5 helps bring in 60S-eIF6; GTP is hydrolized and the IFs are released Met- tRNAimet positioned at the P site
Eukaryotic Translation: Elongation
The key steps in elongation: -Entry of each succeeding aminoacyl-tRNA -Formation of a peptide bond -Movement, or translocation, of the ribosome one codon at a time along the mRNA.
Eukaryotic Translation: Elongation
Elongation factor EF1-GTP brings in the new aminoacyl-tRNA at the A site. Binding of the anticodon with the codon hydrolyzes the GTP of EF1GTP. EF1-GDP is released.
Eukaryotic Translation: Elongation
The 1st peptide bond formation is catalyzed by the peptidyl transferase activity of the large rRNA of the large 60S subunit. The rRNA is a ribozyme. The carboxyl end of the amino acid at the P site is joined to the amino end of the amino acid at the A site to form the peptide bond. Translocation of the ribosome one codon down the mRNA requires hydrolysis of EF2-GTP. Initiator tRNA without the aa is moved to the E site and the 2nd aa-tRNA is moved to the P site.
Eukaryotic Translation: Termination
When the ribosome reaches the stop codon, release factors eRF1 and eRF3GTP enter the A site. eRF1 recognizes all the stop codons. eRFs promote cleavage of the peptide chain from the last tRNA in the P site through hydrolysis of GTP. tRNA and ribosome dissociate. All the factors and subunits are recycled.
The speed of translation
Cells require a lot of proteins. E. coli cells divide every 20 min. Its genome encodes several thousand different proteins (although not all are expressed at the same time). Hundreds and thousands of copies of each protein are usually needed. Translation has to be a very rapid process. Each translation reaction has to be fast: Prokaryotes: 20 aa/sec. Can transcribe and translate all at the same time and place Eukaryotes: 3-5 aa/sec. mRNA processing, traveling, etc. Can have multiple translation processes occurring at once...
Multiple translation events on one mRNA molecule
Polyribosome (or polysome): the complex of mRNA and multiple ribosomes
Eukaryotic circular polysomes
The polyA tail also functions to enhance translation efficiency. PolyA binding protein (PABP) associates with IF4G/IF4E to form a circular mRNA. It allows for a rapid cycling of ribosomes and efficient synthesis of multiple polypeptides form the same mRNA
What about mutations?
STOP codons: UAA, UAG, and UGA What happens when there is a mutation that introduces a new STOP codon that was not previously there? Premature termination codon results in a shorter nonfunctional protein produced by translation
Nonsense mutations
mRNAs with premature STOP codons are detected early by cellular machinery These mRNAs are usually translated only once before being destroyed through nonsense mediated decay (NMD)
How are "nonsense" transcripts detected by the cell?
The Exon-Junction Complex (EJC) is bound 20-24 bp upstream of where two exons have been joined by splicing The EJCs remain bound to the mRNA until displaced by an advancing ribosome during translation What would happen if there was an early STOP codon? How is this mRNA "marked" for destruction?
Molecular chaperones:protein folding
Proteasome Functions to degrade proteins:
Ub
Ub-protein binds to cap
Targeted protein
Ub added by E1, E2, E3 enzymes
Ub removed; protein moves into chamber
subunits degrade protein
The Central Dogma revisited...
prokaryotes and eukaryotic translation
Amino group of the START codon is not formylated in Eukaryotes, but interacts with a soluble initiation factor to enter the P site directly during protein synthesis The initiation complex forms at the 5' end of the mRNA, there is NO Shine-Dalgarno sequence that serves as a recognition site for the ribosome
Some important differences between
Cheat Sheet: Initiation factors
Eukaryotic Initiation Factors (eIF) -The large eIF4 (E,A,G) complex helps to ensure that the 5' end of the mRNA is single-stranded (eIF4E recognizes 5' cap) -eIF2-GTP helps deliver MET-tRNAi to P site -eIF1 associates with 40S subunit -eIF3 and eIF6 help keep subunits apart until associated on mRNA -eIF5 assists bringing together of 40S and 60S subunits