RNA Structure and Processing Flashcards
What is the difference between RNA and DNA nucleotide interactions?
RNA nucleotide interactions are more diverse
What are cellular RNAs processed from?
larger precursors
What generates small stem-loops structures within RNA?
base-pair interactions
What are the 4 nitrogenous bases found in RNA?
- Uracil (pyrimidine)
- Cytosine (pyrimidine)
- Adenine (purine)
- Guanine (purine)
What is a pyrimidine?
a nitrogenous base with one ring
What is a purine?
a nitrogenous base with two rings
What is RNA?
a liner polymer of nucleotides
What is an RNA nucleotide made of?
- ribose sugar
- mono (or more) phosphate(s)
- nitrogenous base
What is found at the 5’ end of an RNA polynucleotide?
a monophosphate group
What is found at the 3’ end of an RNA polynucleotide?
a hydroxyl group
What causes the formation of short helices in RNA polynucleotides?
intramolecular base-pairing interactions within the single stranded RNA
What do stem-loops typically have?
- an irregular structure
- caused by interruptions by non-base-paired nucleotides
What type of base-pairing can occur within RNA polynucleotides?
- canonical (Watson/Crick)
- non-canonical (“wobble” base-pairing)
What are the 3 edges of an RNA nucleotide?
- the Watson/Crick edge
- the Hogsteen (purines) or h/c (pyrimidines) edge
- the sugar edge
What do the 3 edges allow for?
non-canonical base interactions within RNA
How is hydrogen bonding via a nucleotide mediated?
- via ring nitrogen atoms and the attached oxygen atoms or amine groups
- electronegative ring N atoms and ketone O atoms accept H bonds
- H atoms bound to the electronegative ring donate H bonds
What makes up an RNA nucleoside?
a ribose sugar and nitrogenous base
How is RNA synthesised?
- covalently linking the alpha phosphate at the 5’ position of the incoming nucleotide
- with the 3’ hydroxyl group of the ribose unit of another nucleotide
In what direction is the RNA extended?
- 5’ to 3’
- RNA retains a 3’ OH group and a 5’ phosphorylated end
What does the RNA polynucleotide originally have?
- a 5’ triphosphate end
- after processing the RNA typically has a 5’ monophosphate group
How do G-U nucleotides base-pair?
- via the same chemical groups that mediate Watson/Crick base-pairing
- the geometry of the base-pair is unusual
What shape doe heterocyclic bases have?
flat and rigid
Which edges can be involved in noncanonical base-pair interactions within RNA?
the hogsteen edge and the sugar edge
What does non-canonical base-pairing allow RNA to do?
- allows RNA to adopt unique folding patterns
- which are difficult to predict
What are Tetraloops?
- short loops
- containing 4 nucleotides
How are tetraloops stabilised?
through base-staking interactions
What do base-stacking interactions do?
contribute significantly to the stability of nucleic acid helices
What is the Sarcin/Ricin Loop?
- an irregular stem-loop structure
- within large ribosomal RNA
- critical for the process of translation
What is the Function of the Sarcin/Ricin Loop?
- recognised by elongation factor which binds to the ribosome and drives translocation of the ribosome along the mRNA
What is Ricin?
- well known toxic substance
- extremely effective at blocking protein synthesis
- recognises the sarin/ricin loop and removes a single base within the terminal tetra loop structure
How are RNA molecular analogous to proteins?
- have specific 3D shapes
- largely determined by interactions between the nucleotides
What is the secondary structure of RNA?
- a 2D map
- reflects the base-pair interactions which form small helices and stem-loops
What stabilises stem-loops?
- base-pair interactions
- base-stacking interactions
What stabilises small helices?
- ionic base-pairing interactions
- hydrophobic base-stacking interactions
What is coaxial stacking of helices?
- adjacent helices are aligned end to end and stacked on top of each other
- short helices aligned along the same longitudinal axis
What does coaxial stacking allow for?
- a greater degree of base-stacking interaction than seen in 2 separate helices
- contributes to the stability of the folded RNA
Are short helices and stem-loops enough?
- may not provide sufficient thermodynamic stability
- RNA may not fold correctly
Similarities between DNA and RNA helices
geometries are similar
Differences between DNA and RNA helices
- DNA double helix has a broader “major” groove and a narrower “minor” groove
- RNA “major” groove is deeper than DNA’s
What is a “long range” tertiary interaction within RNA?
interactions between sequences which are well separated in the primary sequence of the RNA
What is a “short range” tertiary interaction within RNA?
interactions between closely positioned nucleotides within the primary sequence of the RNA
What is the most common tertiary interaction within RNA?
A-minor motif
What is the A-minor motif?
- 2 adjacent A residues interact with adjacent base-pairs in the minor groove
- through sugar edge interactions
- long range tertiary structure interaction
What are pseudoknots?
- localised tertiary structure interaction
- tightly bound structure
- can be resolved into a single strand by “pulling” the ends apart
How are pseudoknots formed?
- nucleotides within a stem-loop base-pair with nucleotides adjacent to he base of the stem
- two helical elements are coaxially stacked
What does a pseudoknot result in?
tightly folded & compact region of RNA
What type of RNA is predominantly found within a cell?
rRNA
What is the most abundant RNA within cells?
tRNA
How are RNAs generated from primary transcripts?
via processing reactions
What process in the cell do rRNAs and tRNAs function in?
translation
What are the molecular events leading to the production of mature, functional RNA called?
RNA processing reactions
What analysis technique is used to analyse nucleic acids
agarose gel electrophoresis
What type of electrophoresis can shorter nucleic acids be detected on?
- acrylamide gel electrophoresis
- can show rRNA (25S, 18S, 5.8S, 5S) or tRNA
What % does rRNA comprise of cellular RNA?
75-80%
What % does tRNA comprise of cellular RNA?
10%
What % does mRNA comprise of cellular RNA?
5%
What do small nuclear RNAs (snRNA) function as?
pre-mRNA splicing
What do small nucleolar RNAs (snoRNA) function as?
involved in rRNA production
What is an example of a regulatory RNA?
microRNAs
What is cellular RNAs processed from?
- longer precursor molecules
- generated from transcription
How are 3/4 of rRNAs in eukaryotic cells processed?
- via the action of endo- and exonuclease
- cleave RNA within the polynucleotide sequence
- or shorten the RNA from the 5’ to 3’ end
Why is the processing of mRNA transcripts of particular interest?
- sequences are removed through a mechanism not involving RNA cleavage
- additional RNA sequence is typically added in the form of a poly(A) tail
What type of transcripts are eukaryotic mRNA?
monocistronic
What type of transcripts are prokaryotic mRNA?
polycistronic
What is a single polycistronic mRNA translated into?
multiple functionally related proteins
What is a single monocistronic mRNA translated into?
a single polypeptide
What can the up or down regulation of polycistronic mRNAs lead to?
- the coordinated regulation of all the components within a given biological pathway
- in a prokaryote
What modifications are made to eukaryotic mRNAs?
- structural modifications
- the 5’ end is capped by the addition of a single conceded guanosine nucleotide
- the 3’ end is extended by the addition of multiply adenylate residues
What do the structural modifications made to eukaryotic mRNAs do?
- 5’ cap and 3’ poly(A) tail protect the mRNA from degradation by ribonuclease
- stimulate translation of the mRNA
What do polycistronic mRNAs allow for?
- the coordinated expression of all functionally related proteins required for a particular biological pathway
- in one transcript
How are functionally related proteins in eukaryotic cells regulated?
- through regulatory control circuits
- act independently on all the gens required for a particular process
What do ribosomes recognise in oder to start translation?
- nucleotide sequences
- recognised as translation start and stop signals
The translated nucleotide sequence of an mRNA is known as what?
an open reading frame (ORF)
How many open reading frames do polycistronic mRNAs contain?
multiple
How many open reading frames do monocistronic mRNAs contain?
singular one
How are ORFs flanked?
- 5’ leader region
- 3’ untreated region (UTR)
What do the 3’ UTR and 5’ leader region contain?
- important regulatory sequence information
- required for the translation and stability of the mRNA
What do all RNA Polymerase II transcripts receive?
- a noncoded guanosine nucleotide “cap”
- at their 5’ end
How is the 5’ cap attached to the mRNA?
- covalently attached to the 1st transcribed nucleotide
- by a 5’-5’ triphosphate linkage
What is different about the noncoded guanosine nucleotide?
- universally methylated at the N7 position
- referred to as an m7G cap
What modification is observed in most/all eukaryotic mRNAs?
- a sequence of 70 - 200 noncoded A residues
- at the 3’ end
- referred to as a poly(A) tail
What modification is observed in complex eukaryote’s mRNAs?
methylation of the sugar or base unit of the 1st transcribed nucleotide
What is initially required for the termination of RNA Polymerase II transcription?
- a cleavage event
- releasing the mRNA transcript
What is this cleavage event dependent on?
- recognition of specific nucleotide sequences
- found at the end of protein coding genes
Give an example of a recognition sequence required for RNAP II termination?
AAUAAA
What event is coupled to the cleavage reaction of RNAP II termination?
- polyadenylation of the 3’ end
- via the enzyme poly(A) polymerase (PAP)
What does the enzyme poly(A) polymerase do?
- adds contemplated A residues to the 3’ end of a released mRNA transcript
- protecting the RNA from degradation
What are noncoding sequences in the DNA called
introns
How are introns removed from mRNA transcripts to produce complete open reading frames?
pre-mRNA splicing
What carries out pre-mRNA splicing?
- large ribonucleoprotein complex
- called spliceosome
What is involved in the pre-mRNA splicing process?
- 2 transesterification reactions
- the elimination of the intron in the form of a lasso/lariat structure
What catalyses the splicing reaction?
RNA subunits of the spliceosome
What are enzymes containing a catalytic RNA subunit called?
ribozymes
How can the nature of split or interrupted gens be visualised?
electronmicroscopy
Typically how long are introns?
kilobases long
Typically how long are exon sequences?
a few hundred nucleotides long
Approximately what percentage of the primary transcript is intronic?
85%
How is the accuracy of pre-mRNA splicing achieved?
- recognition of conserved 3’ and 5’ splice sites
- structural features of the intron
- the assembly of RNA-binding proteins on sequences found across exons
What are common structural features of introns?
- branch point adenosine
- polypyrimidine tract at the 3’ end
How does pre-mRNA splicing occur?
- via two consecutive transesterification steps
- OH group of an alcohol attacks a phosphodiester linkage
- generating a new phosphodiester linkage
- releasing another OH group
In detail describe the 1st step of the transesterification steps
- 2’ OH group at the branch point Adenosine residue attacks the phosphodiester linkage at the 5’ splice site
- releasing the 5’ exon
- generating a lariat-3’ exon
In detail describe the 2nd step of the transesterification steps
- OH group at the 3’ end of the 5’ exon attacks the phosphodiester linkage at the 5’ end of the 3’ exon
- splicing the 2 exon together
- releasing the intron lariat
What 2 nucleotides are found at the 5’ splice site?
GU
What structural features are found at the 3’ splice sit?
- polypyrimidine tract
- AG nucleotides
What carries out pre-mRNA splicing?
- large ribonucleoprotein complex (RNP)
- called a spliceosome
What is a ribonucleoprotein complex?
a complex containing both RNA and protein
What is the differences between a spliceosome and a ribosome?
- splicesosomes are larger than ribosomes
- splicesosomes are less easily visualised due to their dynamic structure
What are splicesosomes assembled and disassembled on?
pre-mRNA transcripts during the splicing process
What does a spliceosome consist of?
- 5 distinct ribonucleoprotein particles
- each containing a single small nuclear RNA (snRNA) and a small number of proteins
How are splicesosomes positioned correctly on the primary transcripts?
- base pairing interactions between the snRNAs and the pre-mRNA
What happens after splicing has occurred?
- spliced exon and intron lariat is released
- spliceosome is disassembled
What are ribozymes?
enzymes containing an RNA catalytic subunit
What has nuclear pre-mRNA thought to have evolved from?
self-splicing introns
What is another term for small nuclear RNPs (snRNPs)?
snurps
Where does the process of transcription take place in eukaryotes?
the nucleus
Where does the capping of the 5’ end of the transcript in eukaryotes occur?
the nucleus
Where does the polyadenylation of the 3’ end of the eukaryotic transcript occur?
the nucleus
Where does the pre-mRNA splicing in eukaryotes occur?
the nucleus
What type of eukaryotic mRNA transcripts are transported from the nucleus to the cytoplasm?
fully processed, mature mRNA transcripts
When do pre-mRNA processing events typically take place?
- normally are coupled to transcription
- take place cotranscriptionally
How are pre-mRNA processing and transcription coupled?
molecular machineries that carry out mRNA capping, splicing and polyadenylation are recruited to the transcript
What do molecular machineries which carry out pre-mRNA processing interact with?
- RNA polymerase
- at different points along the length of the gene
When does the capping enzyme interact with RNA polymerase?
at the beginning of the gene
When does the cleavage and polyadenylation complex interact with RNA polymerase?
towards the 3’ end of the gene
What mediates the coupling of pre-mRNA processing and transcription?
- changing patterns of post-translational modification of the catalytic subunit of RNA polymerase II
- as the enzyme moves along the gene
What are the 3 main steps in eukaryotic pre-mRNA processing that occurs in the nucleus?
- capping of the 5’ end of the mRNA
- removal of intronic sequences by pre-mRNA splicing
- 3’ end processing (cleavage and polyadenylation)
What do all RNAP II transcripts receive?
m7G cap
What does the catalytic subunit of RNA Polymerase II contain?
an extended C-terminal domain (CTD)
What does the CTD comprise of?
25-50 copies of a serine-rich heptapeptide sequence
How is the CTD domain modified?
- post-transcriptionally
- by the phosphorylation of the serine residues
What happens to the pattern of phosphorylation of the CTD as the RNAPs moves along the gene?
- it changes
- allowing for various complexes required for pre-mRNA processing reactions to bind at different phosphorylated states
- and so are recruited to the polymerase at different times as it moves along the gene
What is the pattern of phosphorylation on the CTD known as?
the CTD code
What is the serine-rich heptapeptide sequence found in the CTD?
YSPTSPS
