Biological roles of RNA
Transmission of genetic info Storage of genetic info (retroviruses) Catalysis and structural components of large macromolecules (ex rRNA and snRNA) Gene regulation (miRNA/siRNA)
What is the mRNA sequence for this gene
5’ACGTCGTCC3’
3’TGCAGCAGG3’
5’ACGTCGTCC3’ the gene, coding strand, sense strand
3’TGCAGCAGG3’ non-coding, antisense, template strand
ACGUCGUCC mRNA
mRNA
- Selected regions are transcribed from DNA to mRNA
- Nucleotide triplets in mRNA (codons) pair with tRNA to code for specific AA
- Prokaryotic mRNA is polycistronic (contains infor for more than one polypeptide chain)
- Eukaryotic mRNA is monocistronic (info for only one polypeptide chain)
- 3’ poly A tail
- 5’ cap of methyl guanosine
tRNA
- 73-93 nucleotides
- Functions in translation of genetic info from mRNA into proteins
- Highly structured
- Acceptor Stem: site where specific AA is linked
- Anticodon loop: Base pairs with codon on mRNA
- Unique nucleotide bases: dihydrouridine, ribothymine, inosine, pseudouridine
rRNA
- Ribosome is 65% protein and 35% protein
- rRNA: 5s, 16s, 23s prokaryotes
- rRNA is the catalytic site of the ribosome
Gene definition and its transcription
A gene is a segment of DNA that functions to generate RNA
Transcribed region of a gene contains the template for RNA synthesis
For mRNA the transcribed region contains the protein coding sequence
Transcription of a gene can proceed in either direction depending on which strand is the template strand
Prokaryotic RNA polymerases
- Synthesize RNA using the template strand of DNA
- Synthesize RNA 5’–>3’ and DONT require a primer
- DO NOT have exonuclease activity
- Core enzyme consis of alpha2betabeta’
- Holoenzyme is core enzyme (RNA Poly) plus sigma
- Different sigma subunits confer promoter specificity
Promoters definition
- Binding sites in DNA where transcription begins
- Regulatory region of DNA generally located upstream of coding sequence (5’ to start of gene)
- Contains DNA consensus (-35 and -10 elements) sequences that are recognized by RNA polymerase
- Consensus sequence is a sequence that is most commonly found in a given region when multiple sequences are aligned
Initiation
- RNA Poly cannot initiate transcription on its own
- Sigma subunit must first bind to the prokaryotic DNA promoter
- Sigma and RNA poly together form a holoenzyme
- Sigma works as a regulatory protein guiding RNA polymerase to specific promoter sequences on the DNA template strand
Initiation continues
Sigma opens the DNA helix, transcription begins
Initiation is complete and the next 3 steps
Sigma releases, mRNA synthesis continues
Elongation
Hairpin forms
Termination
Eukaryotic Transcription more complex because
- Transcription factors instead of sigma factors
- Complex promoters and regulatory regions
- 3 RNA polymerases
Eukaryotic RNA polymerases
- RNA poly I
- preribosomal RNAs (pre-rRNAs): 18s rRNA, 5.8 rRNA, 28 rRNA
- RNA poly II
- mRNAs
- RNA poly III
- tRNAs and 5s rRNA
Eukaryotic RNA processing
- Eukaryotic rRNAs tRNAs and mRNAs are processed (prokaryotic mRNAs arent)
- Primary transcript is the linear, unprocessed RNA
- Eukaryotic mRNA processing takes place in the nucleus prior to mRNA translation in the cytoplasm
- Processing includes
- 5’ capping
- intron splicing
- 3’ polyadenylation
rRNA processing
Preribosomal RNAs are cleaved by ribonucleases to give intermediate sized pieces of rRNA
tRNA processing
- 3’ urasil residues replaced by the CCA sequence found in all mature tRNA
- A 16 nucleotide sequence at the 5’ end is cleaved by RNase P
- A 14 nucleotide intron in the anticodon loop is removed
- Many bases are converted to characteristic modified bases
mRNA processing 3 events
- 5’ capping
- 3’ adenylation
- Intron splicing
5’ mRNA capping
- 5’ capping with a modified guanosine is important for
- Stability of message (protection from ribonucleases)
- Exit from nucleus
- Promoting efficient translation of mRNA to protein
Polyadenylation
- Addition of A’s to 3’ end
- Important for transcription termination
- translation
- mRNA stability
- Nuclear export mRNA
- Requires endonuclease and polyadenylate polymerase
- Pol II synthesizes RNA beyond a cleavage site
- Cleavage signal sequence (AAUAAA) is 20-30 nucletides upstream of cleavage site and is bound by an ezyme complex
Intron Splicing
- Pre-mRNA is spliced to remove introns
- Introns are non-coding intervening sequences
- Zero to dozens of introns in a eukaryotic mRNA
- Range in size 50-20,000 nucleotides
- More DNA devoted to introns than to protein coding exons
Intron Splicing steps
- Occurs in spliceosome
- Spliceosome functions to recognize intron/exon boundaries and cuts/pastes exons
- Splieosome composed of assembled
- small nuclear RNAs (snRNA)
- Small nuclear ribonucleoproteins (snRNP):
- snRNAs complexed with proteins
Alternative Splicing
- Production of two or more distinct proteins from a single protein
- Individual genes express multiple mRNAs (multiple proteins)
- Up to 59% of gene generate multiple mRNAs
- 80% of alternatively spliced mRNAs produce proteins that vary in amino acid sequence
RNA dependent synthesis of RNA and DNA
- Reverse transcriptase
- Synthesizes DNA from viral RNA
- Synthesizes complementary DNA resulting in dsDNA
- Extremely error prone and mutations arise at a high rate, leading to drug resistance
