BMS2062 Lecture Notes - Lecture 3: Sanger Sequencing, Reading Frame, Uridine
30 May 2018
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Week 2. RNA structure and function and, Genome
sequencing
NOT JUST A MESSENGER: RNA ORIGAMI (FUNCTION THROUGH FOLDING)
• ORF > 60 aa is almost certainly part of a gene
• ORFs that are part of authentic genes will be flanked by regulatory motifs which direct
production of RNA
• To make sure sequence encodes for a gene
-> look for ORF
-> look for start/stop codon
-> promoter
• RNA is usually an unstable intermediate between DNA and protein
• Chemical of structure of RNA compared to DNA
o Ribose sugar
o Uracil
o Phosphodiester backbone is the same
o “ae 5 to 3 polaity
• RNA can base pair with DNA or RNA
- in many circumstances, RNA is stably or transiently associated with DNA or other RNA
molecules
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• RNA base pairing:
o Uracil can also base pair with Guanine
o A always pairs with U
• RNA can fold into specific structures:
o ssRNA can form intra-molecular base pairs between inverted repeats to form antiparallel
ds (duplex) structures or stem loops
o unconventional base pairing stabilises the structure
• Elements of RNA structure (via bending, twisting and pairing): distinct shapes eg. kissing hairpin
loop
• Ribosomes are made up of RNA that are folding in complicated structures
-RNA acting as a biological catalyst/machine – capacity to fold up in intricate structures
• Predicting RNA secondary structure
o Several alternative structures can usually be predicted to arise from the same molecule
o The key to identifying the most likely structure is thermodynamics: energy must be
released to form a base paired structure, and the more released, the more stable the
structure
o RNA will fold up into a structure which has the lowest thermodynamic energy = most
stable
o The more negative the G value, the more stable the structure
o Uncommon to find perfect inverted repeat
o Hairpins can form even if inverted repeats are imperfect but each mismatch reduces the
stability of the structure
o Loops are less stable at very small and large sizes, optimum loop size is about 7 bases
• Importance of RNA structure:
o Transcription, in termination, splicing, transport
o Regulation of RNA stability
o Translation ,in initiation, elongation, termination and regulation
o Catalysis
o Types of RNAs produced:
Prokaryotes and eukaryotes: mRNA, rRNA, tRNA, antisense RNA
Eukaryotes only: microRNA
One difference between prokaryote and eukaryote is that eukaryotes require
spliig heeas pokayotes dot
• mRNA:
o has evolved as an expendable information carrier that allows flexibility in how genetic
information is exploited
1. Only genes required are active
2. Amount of gene product produced is proportional to the amount of mRNA
(regulation)
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3. Distinct proteins can be synthesised from a single gene via mRNA modification
(splicing)
• tRNA:
o carries anti-codon that base pairs to the corresponding codon in mRNA
o t‘NA attahed aa is also alled a aioayl o haged t‘NA
• Frame shifting
o Ribosome selects the reading frame by identifying the initiation codon
o Some instances, the ribosome will slip on the mRNA and change frames during
translation
o Slippage is caused by specific mRNA secondary structure
-driven by capacity of RNA to form a stem loop secondary structure
• Differences between prokaryotic and eukaryotic mRNA:
o Nucleus makes a problem for the cell
Eukaryote
Prokaryote
o Transcription and splicing produces single
gene mRNA
o Monocistronic
o Transcription and modification of RNA
occurs in nucleus
o Ribosomes are produced in the nucleolus
o Translation occurs in cytoplasm
o mRNA and ribosomes are actively
transported from nucleus into cytoplasm
o Derived by excision of noncoding segments
(introns) from a precursor mRNA – splicing
o Accurately predicting splice junctions is the
key to gene identification in eukaryotic
genome analyses
o Typical mRNA is reusable – translated more
than once
o Modified at oth 5 ad 3 ed – stable
o 7-ethylguaosie ap is added to 5 ed
o Polyadeylatio stig of As ous at 3
end
o 5 ap ids taslatioal iitiatio fatos
and the small ribosomal subunit
o Transcription without splicing produces multi
gene mRNAs
o Polycistronic
o One time use – translated once before
degradation – degraded as soon as it exits
ribosome
o 5 ad 3 ed ae uodified ad is ot
spliced – very unstable
o Initiation code is preceded by a ribosome
binding site
o Single mRNA can encode several proteins by
incorporating several ribosome binding sites
and ORF in sequence
o Some prokaryotic and many eukaryotic mRNAs have noncoding or untranslated regions
(UTR) at 5 ad 3 eds
o Portions of UTR commonly fold into secondary structures – stems or loops
-> provides binding sites for proteins that stabilise, destabilise or transport mRNA
• Regulation of mRNA
o Eg. iron responsive elements
-Ferritin (iron storage) binds and stores excess cytoplasmic iron
find more resources at oneclass.com
find more resources at oneclass.com
Document Summary
Importance of rna structure: transcription, in termination, splicing, transport, regulation of rna stability, translation ,in initiation, elongation, termination and regulation, catalysis, types of rnas produced: Prokaryotes and eukaryotes: mrna, rrna, trna, antisense rna. Driven by capacity of rna to form a stem loop secondary structure: differences between prokaryotic and eukaryotic mrna, nucleus makes a problem for the cell. Prokaryote: transcription without splicing produces multi gene mrnas, polycistronic. One time use translated once before degradation degraded as soon as it exits ribosome: 5(cid:859) a(cid:374)d 3(cid:859) e(cid:374)d a(cid:396)e u(cid:374)(cid:373)odified a(cid:374)d is (cid:374)ot spliced very unstable. > provides binding sites for proteins that stabilise, destabilise or transport mrna: regulation of mrna, eg. iron responsive elements. Ferritin (iron storage) binds and stores excess cytoplasmic iron. Falls off stem loop: small noncoding micrornas: Can be converted through cleavage into small rna pieces that can bind to protein in cell and act as target sequence.
