Week 2 (Intron splicing) Flashcards
What are exons?
Protein coding sequences of eukaryotic genes
What are introns?
Non protein coding sequences
Main difference between prokaryotic and eukaryotic genetic sequences?
- Bacterial genes typically consist of a continuous stretch of coding DNA that is directly transcribed into mRNA
- Eukaryotic genes contain intervening sequences (introns) thus the coding part of a eukaryotic gene is often only a small fraction of the length of the gene. Both intron and exon sequences are transcribed into RNA
What is the process by which intron sequences are removed?
Intron splicing
What are transesterifications?
Reactions that joins two exons together while removing the intron between them as a ‘lariat’
What could be some benefits of the exon intron arrangement?
- Would facilitate the emergence of new and useful proteins over evolutionary timescales
What is the main advantage of RNA splicing?
- The transcripts of many eukaryotic genes are spliced in more than one way, thereby allowing the same gene to produce a corresponding set of different proteins thus increasing the coding potential of their genome
- enabling genes for new proteins to evolve more easily by the combination of pre-existing genes
Describe the pre-mRNA splicing reaction
- A specific adenine nucleotide in the intron sequence attacks the 5’ splice site and cuts the sugar phosphate backbone of the RNA at this point
- The cut 5’ end of the intron becomes covalently linked to the adenine nucleotide thereby creating a loop in the RNA molecule
- The released 3’OH end of the exon sequence then reacts with the start of the next exon sequence, joining the two exons together and releasing the intron sequence in the shape of a lariat
- The two exon sequences become joined into a continuous coding sequence
- The released intron sequence is eventually broken down into single nucleotides, which are recycled*
What happens to the introns released during splicing?
They eventually break down into single nucleotides which are recycled
What are the specialised RNA molecules that recognise the nucleotide sequences that specify where splicing is to occur?
snRNAs: Small nuclear RNAs U1 U2 U4 U5 U6
what are the invariant nucleotides in the splicing consensus sequence?
GU at the start of the intron and the AG at its end
What is the name of the large assembly of RNA and protein molecules that perform pre-mRNA splicing in the cell?
What is at the core of this assembly?
The spliceosome
snRNPs
How is recognition of the 5’ splice junction, the branch point site, and the 3’ splice junction is performed?
Through base pairing between snRNAs and the consensus RNA sequences in the pre-mRNA substrate
Describe the action of the U1 snRNP
- U1 snRNP forms base pairs with the 5’ splice junction and the BBP (branch point binding protein) and the U2AF (U2 auxiliary factor) recognise the branch point site
Describe the action of the U2 snRNP
u2snRNP displaces BBP and U2AF and forms base pairs with the branch point consensus sequence
Describe the action of the U4/U6.U5 triple snRNP
- In this triple snRNP, the U4 and U6 snRNAs are held firmly together by base-pair interactions
- Subsequent rearrangements break apart the U4/U6 base pairs, allows U6 to displace U1 at the 5’ splice
- This creates the active site that catalyses the first phosphoryl transferase reaction
What completes the splice?
Additional RNA-RNA rearrangements for the active site for the second phosphoryl transferase reaction
What proteins remain on the spliced mRNA molecule?
EJCs (Exon junction complex)
ATP hydrolysis is not required for RNA splicing per se, but what is it required for?
The assembly and rearrangements of the spliceosome
What is the purpose of the arrangements in the spliceosome?
-The allow splicing signals in the pre-RNA to be examined
Allows the spliceosome to check and recheck splicing signals thereby increasing the overall accuracy of splicing
- Secondly the rearrangements that take place in the spliceosome create activation sites for two transesterification reactions
What are EJCs?
exon junction complex
Proteins that mark the site of a successful splicing event
What is ‘exon definition’?
Allows splicing machinery to seek out the relatively homogenously sized exons
What is the function of SR proteins?
They contain a domain rich in serines and arginine’s that assemble on exon sequences and help to mark off each 3’ and 5’ splice site
They also recruit U1 snRNA which marks the downstream exon boundary and U2 which marks the upstream one
SR proteins also bind to splicing enhancers (specific RNA sequences in exons)
Which 3 enzymes are involved in the polyadenylation of eukaryotic pre-mRNA
- CPSF (cleavage and polyadenylation specificity factor)
- CstF (cleavage stimulation factor)
- Poly (A) polymerase
How are cstF and CPSF involved in creating the 3’ end of the mRNA?
They bind to their recognition sequences on the emerging RNA molecule, additional proteins assemble with them to create the 3’ end of the mRNA
How is poly-A-polymerase in polyadenylation?
It adds one at a time approximately 200 A nucleotides to the 3’ end produced by the cleavage
Why isn’t the poly-A directly encoded by the genome?
Unlike other RNA polymerases poly-A Polymerase does not require a template
What helps determine the final length of the Poly-A tail?
Poly-A binding proteins assemble onto the tail
What happens to the newly synthesised RNA that emerges from the polymerase?
It lacks a 5’ cap so it is unprotected therefore is rapidly degraded by a 5’-3’ exonuclease carried along the polymerase tail
How does the cell distinguish from mature mRNA molecules and pre-mRNA?
It can recognise the proteins on specific molecule and when they signify that processing was successfully completed the mRNA is exported from the nucleus into the cytosol where is can be translated in a protein
Give an example of RNA debris
excised (removed) introns
What is the exosome?
A large protein complex whose interior is rich in 3’-5’ RNA exonucleases
What are the functions of hnRNPs (heterogeneous nuclear ribonuclear proteins)?
- Unwind the hairpin helices in the RNA so that splicing and other signals on the RNA can be read more easily
- Package RNA contained in long intron sequences
- Distinguish mature mRNA from the debris left over from RNA processing
What are nuclear pore (complexes)?
Aqueous channels in the nuclear membrane that directly connect the nucleoplasm and the cytosol, small molecules can diffuse freely through these channels
How do macromolecules move through the nuclear pore?
Using nuclear receptors
How do nuclear transport receptors transport mRNA molecules?
Moves the mRNA through the nuclear pore complex then dissociates and re enters the nucleus where it can be used again
What does polymerase I lack?
What does this explain about its transcripts?
a C-terminal tail
helps to explain why its transcripts are neither capped or polyadenylated
Are all rRNA molecules transcribed together?
3 out of the 4 are made by chemically modifying and cleaving a single large precursor rRNA; the fourth is synthesized by a cluster of genes by polymerase III and does not require chemical modification
Describe the function of snoRNAs (Small nuclear RNAs)
Promote cleavage of the precursor rRNA into the mature rRNA by causing conformational changes (chemical modifications) that expose these sites to nucleases
They perform their functions in the nucleolus
In short they bring RNA modifying enzymes to the appropriate position by base pairing to complementary sequences in the precursor rRNA
What are snoRNPs (small nuclear ribonuclear proteins)?
snoRNAs bound to proteins
They contain both the guide sequence and the enzymes that modify the rRNA
Unlike many major organelles, the nucleolus is not membrane bound, instead has as huge aggregate macromolecules:
rRNA genes, precursor rRNAs, mature rRNAs, rRNA processing enzymes, snoRNPs, ribosomal proteins, partly assembled ribosomes etc
What does the size of the nucleolus indicate?
The number of ribosomes that the cell is producing
How is the U6 snRNP produced?
U6 snRNA is chemically modified by snoRNAs in the nucleolus before it is assembled into the U6 snRNP
Where are the genes encoding tRNAs clustered?
In the nucleolus
What is the large factory at which different noncoding RNAs are transcribed, processed and assembled with proteins to form a large variety of ribonucleoprotein complexes?
The nucleolus
What are Cajal bodies?
Cajal bodies are sites where the snRNPs and snoRNPs undergo their final maturation steps, and where the snRNPs are recycled and their RNAs are “reset” after the rearrangements that occur during splicing
Give a summary of bacterial transcription
Bacteria contain a single type of RNA polymerase (the enzyme that carries out the transcription of DNA into RNA). An mRNA molecule is produced after this enzyme initiates transcription at a promoter, synthesizes the RNA by chain elongation, stops transcription at a terminator, and releases both the DNA template and the completed mRNA molecule.
Give a summary of eukaryotic transcription
- In eukaryotic cells, the process of transcription is much more complex, and there are three RNA polymerases—polymerase I, II, and III—that are related evolutionarily to one another and to the bacterial polymerase.
- RNA polymerase II synthesizes eukaryotic mRNA. This enzyme requires a set of additional proteins, both the general transcription factors, and specific transcriptional activator proteins, to initiate transcription on a DNA template.
- It requires still more proteins (including chromatin remodeling complexes and histone-modifying enzymes) to initiate transcription on its chromatin templates inside the cell. During the elongation phase of transcription, the nascent RNA undergoes three types of processing events: a special nucleotide is added to its 5ʹ end (capping), intron sequences are removed from the middle of the RNA molecule (splicing), and the 3ʹ end of the RNA is generated (cleavage and polyadenylation). Each of these processes is initiated by proteins that travel along with RNA polymerase II by binding to sites on its long, extended C-terminal tail.
- Splicing is unusual in that many of its key steps are carried out by specialized RNA molecules rather than proteins.
- Only properly processed mRNAs are passed through nuclear pore complexes into the cytosol, where they are translated into protein.
What happens to the RNA that is the final product of genes rather than proteins
- In eukaryotes, these genes are usually transcribed by either RNA polymerase I or RNA polymerase III. RNA polymerase I makes the ribosomal RNAs.
- After their synthesis as a large precursor, the rRNAs are chemically modified, cleaved, and assembled into the two ribosomal subunits in the nucleolus—a distinct subnuclear structure that also helps to process some smaller RNA–protein complexes in the cell.
- Additional subnuclear structures (including Cajal bodies and interchromatin granule clusters) are sites where components involved in RNA processing are assembled, stored, and recycled.
- The high concentration of components in such “factories” ensures that the processes being catalyzed are rapid and efficient.
RNA is a linear polymer of four different nucleotides, how many possible combinations are there of the 3 nuceotides?
4x4x4=64
Why is the genetic code said to be redundant?
some amino acids are specified by more than one triplet
What is a codon?
A group of three consecutive nucleotides in RNA specifies either one amino acid or a stop to the translation process.
The genetic code is used universally in all present-day organisms. Although a few slight differences in the code have been found:
- These are chiefly in the DNA of mitochondria.
- Mitochondria have their own transcription and protein-synthesis systems that operate quite independently from those of the rest of the cell
What is the function of transfer RNAs (tRNAs)?
- The codons in an mRNA molecule do not directly recognize the amino acids they specify: the group of three nucleotides does not, for example, bind directly to the amino acid.
- Rather, the translation of mRNA into protein depends on tRNA molecules that can recognize and bind both to the codon and, at another site on their surface, to the amino acid.
What is an anticodon?
- The sequence of three nucleotides that base-pairs with a codon in mRNA
- The amino acid matching the codon/anticodon pair is attached at the 3ʹ end of the tRNA.
Why do tRNA molecules contain some unusual bases?
They are produced by chemical modification after the tRNA has been synthesized
Describe and explain the shape of a tRNA molecule?
- Four short segments of the folded tRNA are double-helical, producing a molecule that looks like a cloverleaf when drawn schematically
- For example, a 5ʹ-GCUC-3ʹ sequence in one part of a polynucleotide chain can form a relatively strong association with a 5ʹ-GAGC-3ʹ sequence in another region of the same molecule.
- The cloverleaf undergoes further folding to form a compact L-shaped structure that is held together by additional hydrogen bonds between different regions of the molecule
Which two regions of unpaired nucleotides situated at either end of the L-shaped molecule are crucial to the function of tRNA in protein synthesis?
- A short single-stranded region at the 3ʹ end of the molecule
- One of these regions forms the anticodon
Which end of the tRNA is the amino acid matching the codon attached?
The 3’ end
What does the redundancy of amino acids imply?
- there is more than one tRNA for many of the amino acids
or
-tRNA molecules can base-pair with more than one codon.
What is wobble base pairing and what does it imply?
- accurate base-pairing only at the first two positions of the codon and can tolerate a mismatch at the third of the codon position
- This wobble base-pairing explains why so many of the alternative codons for an amino acid differ only in their third nucleotide
How is inosine formed?
formed from the deamination of adenosine
What is the main difference between the base pairing that occurs at position 1 and 22 compared with that of the wobble position?
They are generally weaker than conventional base pairs.
Codon–anticodon base-pairing is more stringent at positions 1 and 2 of the codon, where only conventional base pairs are permitted
How does tRNA splicing differ from mRNA splicing?
rather than generating a lariat intermediate, tRNA splicing uses a cutand-paste mechanism that is catalyzed by proteins
What is the quality-control steps in the generation of tRNAs?
-Trimming and splicing both require the precursor tRNA to be correctly folded in its cloverleaf configuration. -Because misfolded tRNA precursors will not be processed properly, the trimming and splicing reactions serve as quality-control steps in the generation of tRNAs.
How often does tRNA modification occur?
—nearly 1 in 10 nucleotides in each mature tRNA molecule is an altered version of a standard G, U, C, or A ribonucleotide
What is the purpose of tRNA modification?
- affect the conformation and base-pairing of the anticodon and thereby facilitate the recognition of the appropriate mRNA codon by the tRNA molecule
- Others affect the accuracy with which the tRNA is attached to the correct amino acid
What is the function of aminoacyl-tRNA synthetases?
- Recognition and attachment of the correct amino acid
- They covalently couple each amino acid to its appropriate set of tRNA molecules
one attaches glycine to all tRNAs that recognize codons for glycine, another attaches alanine to all tRNAs that recognize codons for alanine, and so on.
Explain Amino acid coupling in prokaryotes?
- Many bacteria, however, have fewer than 20 synthetases, and the same synthetase enzyme is responsible for coupling more than one amino acid to the appropriate tRNAs.
- In these cases, a single synthetase places the identical amino acid on two different types of tRNAs, only one of which has an anticodon that matches the amino acid
- A second enzyme then chemically modifies each “incorrectly” attached amino acid so that it now corresponds to the anticodon displayed by its covalently linked tRNA
Explain Amino acid activation by synthetase enzymes
- The energy of ATP hydrolysis is used to attach each amino acid to its tRNA molecule in a high-energy linkage.
- The amino acid is first activated through the linkage of its carboxyl group directly to AMP, forming an adenylated amino acid; the linkage of the AMP, normally an unfavourable reaction, is driven by the hydrolysis of the ATP molecule that donates the AMP.
- Without leaving the synthetase enzyme, the AMP-linked carboxyl group on the amino acid is then transferred to a hydroxyl group on the sugar at the 3ʹ end of the tRNA molecule.
- This transfer joins the amino acid by an activated ester linkage to the tRNA and forms the final aminoacyl-tRNA molecule.
Explain the structure of the aminoacyl-tRNA linkage
The carboxyl end of the amino acid forms an ester bond to ribose. Because the hydrolysis of this ester bond is associated with a large favourable change in free energy, an amino acid held in this way is said to be activated
How does synthetase enzymes select the correct amino acid?
- The correct amino acid has the highest affinity for the active-site pocket of its synthetase and is therefore favoured over the other 19; in particular, amino acids larger than the correct one are excluded from the active site
Explain how the genetic code is translated by means of two adaptors that act one after another
The first adaptor is the aminoacyl-tRNA synthetase, which couples a particular amino acid to its corresponding tRNA; the second adaptor is the tRNA molecule itself, whose anticodon forms base pairs with the appropriate codon on the mRNA. An error in either step would cause the wrong amino acid to be incorporated into a protein chain
Describe the second discrimination step that ensure that an aminoacyl-tRNA synthetase links the correct amino acid to each tRNA
- A second discrimination step occurs after the amino acid has been covalently linked to AMP when tRNA binds, the synthetase tries to force the adenylated amino acid into a second editing pocket in the enzyme.
- The precise dimensions of this pocket exclude the correct amino acid, while allowing access by closely related amino acids. In the editing pocket, an amino acid is removed from the AMP (or from the tRNA itself if the aminoacyl-tRNA bond has already formed) by hydrolysis.
How do tRNA synthetases directly recognize the matching tRNA anticodon?
- These synthetases contain three adjacent nucleotide-binding pockets, each of which is complementary in shape and charge to a nucleotide in the anticodon.
- For other synthetases, the nucleotide sequence of the amino acid-accepting arm (acceptor stem) is the key recognition determinant.
- In most cases, however, the synthetase “reads” the nucleotides at several different positions on the tRNA.
What is the fundamental reaction of protein synthesis?
The formation of a peptide bond between the carboxyl group at the end of a growing polypeptide chain and a free amino group on an incoming amino acid
-Consequently, a protein is synthesized stepwise from its N-terminal end to its C-terminal end. Throughout the entire process, the growing carboxyl end of the polypeptide chain remains activated by its covalent attachment to a tRNA molecule
Describe The incorporation of an amino acid into a protein
- A polypeptide chain grows by the stepwise addition of amino acids to its C-terminal end.
- The formation of each peptide bond is energetically favorable because the growing C-terminus has been activated by the covalent attachment of a tRNA molecule. (each amino acid added carries with it the activation energy for the addition of the next amino acid rather than the energy for its own addition)
- The peptidyl-tRNA linkage that activates the growing end is regenerated during each addition
Where are ribosomes synthesised?
- The large and small ribosome subunits are assembled at the nucleolus, where newly transcribed and modified rRNAs associate with the ribosomal proteins that have been transported into the nucleus after their synthesis in the cytoplasm.
- These two ribosomal subunits are then exported to the cytoplasm, where they join together to synthesize proteins.
When does pre-mRNA become mRNA?
When 5’ and 3’ end processing and splicing has taken place
What 3 portions of the precursor RNA molecule must be recognised by the splicing machinery?
- 5’ splice site
- 3’ splice site
- the branch point in the intron sequence that forms the base of the excised lariat
What are many of the y steps in RNA splicing performed by?
RNA molecules rather than proteins
What is the function of the spliceosome?
The spliceosome recognises the splicing signals on a pre-mRNA molecule, brings the two ends of the intron together, and provides the enzymatic activity for the two reaction steps required
What are the last 3 bases in tRNA in the 5’ to 3’ direction before the amino acid?
CCA
Which end of the tRNA molecule does amino acyl tRNA synthetase catalyse a covalent link?
The 3’ end
How many tRNA molecules are transcribed together (in prokaryotes)?
between 1 and 7
How many tRNA synthetase in E.coli for each amino acid?
1
Which end of the tRNA molecule is the amino acid attached?
Finally, the correct amino acid
is attached to the sequence 5’-CCA-3’, which is always present at the 3’ end of the tRNA molecule. This process is called aminoacylation (or charging) and the mature tRNA is now ready to take part in translation.
What sequence 5’-3’ comes at the end (just before) amino acid attached to a tRNA molecule?
CCA
Is ATP hydrolysis required for the chemistry for the RNA splicing?
- The two transesterification reactions preserve high energy phosphate bonds
- However, extensive ATP hydrolysis is required for the assembly and rearrangements of the spliceosome
What happens as splicing proceeds?
The base pairs are broken and U1 is laced by U6
What helps to increase the overall accuracy of splicing?
RNA-RNA rearrangement ( the formation of one RNA-RNA interaction requires the disruption of another)
How are RNA rearrangements related to transesterificstion reactions?
The rearrangements that take place in the spliceosome create the active sites for the two transesterificstion reactions
What happens after splicing chemistry complete?
The snRNPs remain bound to the lariat
What is required for the disassociation of snRNPs from the lariat?
Another series of RNA-RNA rearrangements that require ATP hydrolysis, thereby returning the snRNAs to their original configuration
What happens at the completion of a splice?
The spliceosome directs EJCs t the mRNA near the position formerly occupied by the intron
