RNA Synthesis Notes

Ribonucleic Acids

• Major Classes & Chemical Structure:

  • mRNA (messenger RNA): Carries genetic information from DNA to ribosomes for protein synthesis.

  • tRNA (transfer RNA): Transfers amino acids to the ribosome for protein synthesis, based on mRNA codons.

  • rRNA (ribosomal RNA): Forms the structural and catalytic core of ribosomes, essential for protein synthesis.

• Synthesis of RNA:

  • Transcription: Process by which RNA is synthesized from a DNA template.

  • Post-transcriptional modifications: Modifications to RNA after transcription, including splicing, capping, and polyadenylation.

RNA Basics

• RNA is a sequence of ribonucleoside monophosphates (A, G, C, U).

• Location: Nucleolus, nucleus, cytoplasm, ribosomes

• Contains many unusual and modified bases.

RNA Types & Percentages

• Ribosomal RNA (rRNA): 80-90%

• Messenger RNA (mRNA): 2-5%

• Transfer RNA (tRNA): 10-15%

• Small RNA:

  • snRNA (small nuclear RNA - nuclear): Involved in splicing mRNA precursors.

  • snoRNA (small nucleolar RNA - nucleolar): Guides chemical modifications of other RNAs (tRNA, rRNA, snRNA).

  • scRNA (small cytoplasmic RNA - conditional)

  • miRNA (micro RNA): Regulates gene expression by binding to mRNA.

  • siRNA (small interfering RNA - interfering): Triggers RNA interference, blocking gene expression.

RNA Viruses

• Can be ssRNA (single-stranded RNA) or dsRNA (double-stranded RNA).

Messenger RNA (mRNA)

• Function: Carries genetic information from nucleus to cytoplasm.

• Role: Serves as template for protein synthesis (translation).

• Synthesis: Created on a DNA molecule by transcribing one strand in the nucleus.

• Heteronuclear RNA (hnRNA):

  • Primary transcript.

  • Contains exons and introns.

  • Undergoes splicing and processing of ends to become mature mRNA.

mRNA Structure & Features

• 5' Cap: Modified guanine nucleotide added to the 5' end.

• 5'-untranslated region (30-300 nucleotides): Region before the start codon.

• Start codon (AUG): Initiates translation.

• Coding sequence (300 - 3,000 nucleotides): Sequence that codes for the protein.

• Stop codon (UAG, UAA, UGA): Terminates translation.

• 3'-untranslated region (20-250 nucleotides): Region after the stop codon.

• PolyA tail (30-300 adenine nucleotides): String of adenine nucleotides added to the 3' end.

mRNA - The CAP Structure

• Consists of 7-methylguanine nucleotide added to the 5′-end of the mRNA by an unusual 5'-5'-triphosphate linkage.

• Types: CAP 0, CAP 1, CAP 2

• Critical for recognition and proper attachment of mRNA to the ribosomes.

• Important for protection of mRNA from 5'-exonucleases.

mRNA - CAP Structure details

• 5'-5' phosphodiester bond

• Formulas:

  • CAP 0: $m^7GpppXpYp$

  • CAP 1: $m^7GpppXmpYp$

  • CAP 2: $m^7GpppXmpYmp$

mRNA - Coding Sequence

• Composed of codons (triplets of bases) specifying particular amino acids.

• Start codon: AUG (codes for methionine), site of translation initiation.

• Stop codons: UAA, UAG, UGA; signals for translation termination (do not specify any amino acid).

mRNA - Untranslated Regions (UTRs)

• Sequences of the mRNA that are not translated to protein primary structure.

• Types: 5'-UTR and 3'-UTR

• Functions: mRNA stability, mRNA localization, efficiency of translation.

mRNA - PolyA Tail

• Sequence of 80-250 adenine nucleotides.

• Functions: Important for export from the nucleus, translation initiation, protection against degradation of mRNA.

Transfer RNA (tRNA)

• Essential role during translation.

• Decodes genetic code through base pairing of its anticodon with mRNA codon.

• Transfers amino acid, specific to particular codon according to genetic code.

• Clover-leaf structure:

  • Containing 4 different loops (D loop, Anticodon loop, TψC loop, Variable loop).

  • Characterised by presence of unusual/modified bases (dihydrouridine, 2'-O-methylguanidine, 7-methylguanidine, pseudouridine).

• Canonic sequence: 5'-CCA-3' at the 3'-end is important for amino acid binding.

tRNA - Structure Details

• 3'-end: Acceptor site (CCA)

• D loop: Contains dihydrouridine

• Anticodon loop: Contains anticodon

• TψC loop: Contains pseudouridine

• Variable loop: Varies in size and is used for classification.

Ribosomal RNA (rRNA)

• Complicated secondary and tertiary structure.

• Types: 5S, 5.8S, 18S, and 28S rRNA; mitochondrial 12S and 16S rRNA

• Structural and functional components of ribosomes where they complex with ribosomal proteins.

• Two precursors: 5S-rRNA, 45S-rRNA

Ribosomes

• Prokaryotic Ribosome: 70S (50S + 30S subunits; 21 & 31 proteins respectively)

• Eukaryotic Ribosome: 80S (60S + 40S subunits; 30 & 40 proteins respectively)

Small Nuclear RNA (snRNA)

• Involved in the splicing of mRNA precursors.

• Make complexes with proteins called spliceosomes.

• Three double-stranded regions connected by three loops and additional double-stranded region connecting branched part to linear part containing additional loop.

• 100 – 300 nucleotides.

Micro RNA (miRNA)

• Short RNA (~22 nucleotides).

• Involved in regulation of gene expression.

• Binds to the complementary sequence of particular mRNA, causing either degradation of mRNA or inhibition of translation.

Transcription

• RNA synthesis, transfer of genetic information from DNA to RNA.

• DNA noncoding strand is template for RNA synthesis.

• RNA contains uracil instead of thymine; complementarity during transcription A ― U.

• RNA polymerase: catalyses the synthesis of RNA, ATP, CTP, GTP, UTP, $Mg^{2+}$, and other proteins.

RNA Polymerases

• RNA polymerase I: in the nucleolus, synthesises precursor rRNAs.

• RNA polymerase II: in the nucleus, synthesizes heteronuclear RNA (hnRNA → mRNA), small nuclear RNA.

• RNA polymerase III: in the nucleus, synthesizes precursors of tRNA, 5S rRNA, small RNAs.

• Mitochondrial RNA polymerase: transcription in mitochondria.

Promoter

• A region of DNA that initiates the transcription of a particular gene.

• Located before the gene, acts as a signal for the RNA polymerase and transcription factors.

• Direction of RNA polymerase: 3' → 5'

• Direction of elongated RNA: 5' → 3'

• Phosphodiester bond: C3' and C5'

• Termination: termination of RNA synthesis, terminator = terminational signal = stop signal.

Biochemical Principle of Transcription

• ribonucleoside triphosphate + $\text{RN}<em>n \rightarrow \text{RN}</em>{n+1}$ + pyrophosphate

• Catalyzed by RNA polymerase, requires $Mg^{2+}$ and DNA template.

• Involves elongated RNA strand and pyrophosphatase.

Prokaryotic Promoter

• -35 region (recognition sequence TTGACA)

• -10 region (Pribnow box TATAAT)

• +1/start point

Prokaryotic Promoter - Sigma Factor.

• A σ factor: is a protein needed for initiation of transcription in bacteria.

• It is binding to sequence called recognition sequence (TTGACA).

• The σ factor, together with RNA polymerase, is known as the RNA polymerase holoenzyme.

• Pribnow box (TATAAT): facilitates the localized DNA unwinding.

Transcription - Steps

• INITIATION

  • Binding of σ factor on promoter region of DNA

  • Binding of RNA polymerase

• ELONGATION

  • Nucleoside triphosphate (ATP, CTP, GTP, UTP)

  • Synthesised RNA

• TERMINATION

  • Release of RNA polymerase and σ factor

• Product of transcription - RNA

Eukaryotic Promoter

• Enhancer: Enhances transcription.

• Silencer: Silences transcription.

• GC region (-200 to -40 region)

• CAAT box (-32 to -16 region)

• TATA box (Goldberg-Hogness box)

• +1/start point

Eukaryotic Promoter - Basal Transcription Factors

• The initiation of transcription in eukaryotes; they are found -90, -80 and -30 bp upstream from transcription start site

• TATA box: the most characterised type of core promoter; TATAAAA; -30 to -25 bp upstream

• CAAT box: the second important element; -80 bp; GGCCAATCT

• Transcription starts with binding of TATA-binding protein (TBP; part of transcription factor II D – TFBIID) to the TATA box.

Eukaryotic Promoter - Transcription Factor Binding

• After TFIID binds to the TATA box via the TBP, five more transcription factors (TFIIA, TFIIB, TFIIF, TFIIE, and TFIIH) and RNA polymerase II combine around the TATA box.

• A preinitiation complex: TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, and RNA polymerase II

Transcription - Elongation

• Same comparing prokaryotes and eukaryotes

• Messenger RNA transcription involves often multiple RNA polymerases II on a single DNA template and multiple round of transcription.

• In eukaryotes, early during elongation, the 5'–end of pre-mRNA are modified by addition of 7-methyl guanosine caps.

Transcription - Termination

• Prokaryotes:

  • RNA transcription stops when the newly synthesized RNA molecule forms a GC rich hairpin loop followed by a stretch of uracils.

  • A binding of a specific protein called ρ-factor to specific sequences destabilizes the interaction between the template and the mRNA, thus releasing mRNA.

• Eukaryotes:

  • Less understood but involves cleavage of the synthesized transcript followed by template-independent addition of stretch of adenosine at its 3'-end.

Post-Transcriptional RNA Processing

• Transcription: DNA → primary transcripts of RNAs → processing (maturation) RNA

• rRNA: 45S-rRNA → 18S, 5.8S and 28S rRNA

• tRNA: pre-tRNA → 50 different tRNAs

• mRNA: hnRNA → mRNA for each protein.

Post-Transcriptional RNA Processing - Details

• rRNA: 45S pre-rRNA is processed into 18S, 5.8S, and 28S rRNA, which combine with proteins to form ribosomes (40S and 60S subunits, ultimately forming the 80S ribosome).

• tRNA: Pre-tRNA undergoes cleavage of introns, ligation, modification of bases, and adding of CCA at the 3' end.

• mRNA: hnRNA undergoes capping, methylation, polyadenylation, splicing out of introns, and ligation of exons to form mature mRNA with 5'-UTR and 3'-UTR.

Post-Transcriptional RNA Processing - miRNA

• DNA-miRNA gene -

• TRANSCRIPTION -

• Pri-miRNA -

• Drosha -

• Pre-miRNA -

• Dicer -

• Export from nucleus -

• Mature miRNA

Reverse Transcription

• Transfer information form RNA to DNA

• Reverse transcriptase: viral RNA is converted to DNA in the host, and complementary DNA (cDNA) is formed.

• The RNA is depredated, and the second DNA strand is synthesized.

• ds-cDNA: integration into the host DNA

• Human immunodeficiency virus (HIV): has reverse transcriptase and causes the disease AIDS.

Reverse Transcription - Details

• RNA + Reverse transcriptase + dNTPs → cDNA

• RNAase H activity of reverse transcriptase degrades RNA

• DNA polymerase synthesizes the second DNA strand → ds cDNA.

RNA as Enzyme

• A RIBOZYME: is a type of RNA with catalytic