Essential Molecular Biology (BIOC0007) Notes - RNA

RNA – Summary
RNA Transcription
 RNA transcription – summary (DNA  RNA  protein)
o Transcription
 Copying of coding strand of DNA (sense strand) into an RNA molecule (transcript)
 RNA-transcript = identical to coding strand – but uses uracil instead of thymine
 DNA is transcribed to RNA – by RNA polymerase
 Link nucleotides  to form an RNA strand – using DNA strand as a template
o Phosphodiester bond between 3’ OH of growing RNA strand + 5’ phosphate
of incoming rNTP
o RNA molecules must be processed after transcription
 Spliced  have a 5’ cap and poly-A-tail on their heads
o Translation
 mRNA sequence is read by a ribosome – to produce a protein
 RNA polymerase
o Main enzyme involved in transcription
o Uses a single-stranded DNA template – to synthesise a complementary
strand of RNA
 Builds an RNA strand in the 5’ to 3’ direction
 Adding each new ribonucleotide (rNTP) to the 3’ end of
the strand (rNTP = ribonucleotide tri-phosphate)
o Prokaryotic vs eukaryotic RNA polymerases
 Differences
 Prokaryotes have 1 RNA polymerase
 Eukaryotes have 3 RNA polymerase
 Made up of different subunits
 Similarities
 RNA polymerase is a large multi-subunit molecule
 Transcribes DNA to RNA without needing a primer
 Has a highly conserved structure = core enzyme – responsible for process of
synthesis + relies on extra proteins
o Structure
 Prokaryotic RNA polymerase
 Smallest
 Complex of 400kD
 Made of 5 subunits
o 2 copies of α subunit – scaffolding
rule
o 1 β subunit = active site where
rNTPs bind
o 1 β’ subunit = DNA binding
o 1 ω subunit = unknown role
 Subunits assemble to form a large complex with duel-like lobes – separated by a
cleft
o Enzyme active site
 At the base of the cleft + accessible through opening in β’ subunit =
2 points of access to active site
 In some bacteria – β and β’ subunits are produced as a single polynucleotide
 Eukaryotic RNA polymerases
 3 eukaryotic polymerases
o R1, R2, R3

, RNA – Summary
 Contain more subunits than RNA polymerases
o Ranging from 12-17 – depending on polymerases (Polymerase II = 12)
 Stages of transcription
o Initiation
 RNA polymerase binds to promotor  separates the DNA strands  providing single-
stranded template needed for transcription
o Elongation
 Anti-sense strand = template for RNA polymerase
 RNA polymerase reads the template (non-coding strand) one base at a time –
building an RNA molecule using complementary nucleotides  resulting in a chain
growing from 5’ to 3’
 RNA transcript carries the same information as the non-template (sense) strand of DNA
 Contains uracil (U) instead of thymine (T)
o Termination
 Terminators = sequence of DNA that signals the RNA transcript is complete
 Once transcribed = cause the transcript to be released from the RNA polymerase
 Eukaryotic RNA modifications
o In bacteria
 RNA transcripts can act as mRNAs (messenger RNAs) straight away
o In eukaryotes
 Pre-mRNA = transcript of a protein-coding gene needs extra processing before translation
 Have their ends modified – addition of a 5’ cap at the beginning + 3’ poly-A-tail at
the end = increases the stability of mRNA
 Undergo splicing – introns are removed + exons are joined back together
o Gives the mRNA its correct sequence
 Promotor recognition – differs in prokaryotes and eukaryotes
o Locating promotor
 RNA polymerase cannot locate the start of transcription (+1) on their own
 Additional proteins guide core enzyme complex to promotor region
 Promotor = sequence of DNA adjacent to transcription start site near the beginning
of the gene where RNA polymerase binds – specific to each gene
 Prokaryotes = sigma factor
 Eukaryotes = transcription factor
o In prokaryotes – sigma factor
 RNA polymerase needs sigma factor (enzyme) = directly contacts promotor sequence –
ensuring correct start for transcription
 Sigma factor subunit + RNA polymerase = holoenzyme
o Sigma factor binds to specific sequences upstream of transcription site –
guiding the RNA polymerase to the promotor
 Bacteria have a variety of sigma factors = recognise different
promotor sequences – allowing cell to alter gene expression
 Sigma 70 = common factor in E.coli – responsible for the
initiation of most genes
 Bacterial promotors
 Contain 2 key recognition elements
o Conserved sequence at -35bp
o Conserved sequence at -10bp
 Spatiotemporal gene expression
o There are differences in spacing between conserved sequences – affecting
the binding affinity of different sigma factors
 Variations contribute to the spatiotemporal gene expression