Transcription: DNA to RNA

• Transcription is the process of producing RNA from a DNA template

• Dr. Rachel Hunt can be contacted at [email protected]

• Eukaryotes send instructions to the cytoplasm which vary by cell/tissue type

• The central dogma of molecular biology involves transcription (DNA to RNA) and translation

DNA versus RNA

• DNA is double-stranded, while RNA is single-stranded, though it can form secondary structures
• RNA contains ribose, having a hydroxyl group (OH) at the 2' position, unlike DNA's deoxyribose
• DNA uses the bases ACGT, while RNA uses ACGU, with uracil replacing thymine

Types of RNA

• Messenger RNA (mRNA) encodes proteins

• Ribosomal RNA (rRNA) forms structural and enzymatic components of ribosomes

• Transfer RNA (tRNA) delivers amino acids to the ribosome

• All RNAs are produced by transcription

• mRNA and tRNA are involved in translation

• mRNA is produced from a DNA template strand

mRNA Transcription

• Transcription starts at a specific site
• The 5' UTR contains signals for translation initiation
• The 3' UTR contains translational termination signals
• The polymerization of ribonucleoside triphosphates into RNA is facilitated by RNA polymerase

E. Coli RNA Polymerase

• E. coli RNA polymerase uses ribonucleoside triphosphates to synthesize RNA
• RNA polymerase unwinds DNA as it moves down the strand
• Sigma factors dictate where transcription will occur

Part A Summary

• RNA differs from DNA by being single-stranded, containing ribose, and using uracil instead of thymine
• mRNA encodes proteins, rRNA is a structural component of ribosomes, and tRNA delivers amino acids
• Transcription produces mRNA from a DNA template
• RNA polymerase polymerizes ribonucleoside triphosphates into RNA

Prokaryotic Transcription

• Transcription has three primary steps: initiation, elongation, and termination
• Short recognition sequences in the promoter region align RNA polymerase at the transcription start site

Initiation

• A 'sigma factor' binds to the -10 (Pribnow) and -35 boxes, recruiting RNA polymerase to the transcription start site

Elongation

• RNA polymerase unwinds DNA, synthesizes mRNA by adding complementary nucleotides, requires no primer
• Sigma factor dissociates after the first few nucleotides are added
• Nucleotides are added in the 5' to 3' direction during this process
• mRNA sequence mirrors the non-template (coding) strand of DNA
• Nucleotides in RNA are joined by covalent phosphodiester bonds

Termination

• Termination is controlled by GC-rich stop sequences in DNA

Rho independent termination

• Rho independent termination is the most common method of transcription termination in E. coli
• mRNA transcript forms a stem loop structure
• This reduces binding of mRNA to the DNA template
• Followed by a string of weak A-U pairings on the template

Rho dependent termination

• Involves RNA polymerase pausing at GC-rich sequences
• Rho factor, possessing helicase activity, unwinds the DNA-RNA duplex separating nascent RNA

Prokaryotic Genes

• In a hypothetical prokaryotic genome segment, one can determine specific sequence functions
• Sigma factors bind to the -10 (Pribnow) and -35 boxes, positioning RNA polymerase
• Transcription begins at nucleotide 98 and promoter region spans ~nucleotides 58 and 97
• The U rich sequence forms stem loop for transcription
• 5' untranslated region (5' UTR) green
• 3' untranslated region (3' UTR) blue
• Transcript length is about 612 nucleotides

Part B Summary

• Transcription consists of initiation, elongation, and termination
• Sigma factor binds promoter sequences, positioning RNA polymerase
• RNA polymerase unwinds DNA during elongation, synthesizing mRNA
• Nucleotides are added in the 5' to 3' direction
• Prokaryotic transcription is terminated via rho-dependent and rho-independent

Eukaryotic Transcription

• Eukaryotes have 3 RNA polymerases: RNA Pol I (rRNA), RNA Pol II (mRNA, snRNA), RNA Pol III (5S rRNA, tRNA)
• RNA Pol II contains a C-terminal domain (CTD) 'tail'

C-Terminal Domain

• The C-terminal domain (CTD) recruits processing factors to eukaryotic mRNAs
• Only fully processed mRNA can be exported for translation
• The primary transcript is called heterogeneous nuclear RNA (hnRNA) or pre-mRNA

Co-transcriptional/post-transcriptional processing in Eukaryotes

• 7-methylguanosine cap is formed at the 5' end, protects
• Guanosine base is added ‘by reaction’ with GTP
• The transcript protects export /translation from degredation

Polyadenylation

• (50-250) adenosine (A) residues added to 3' end of mRNA, not encoded by the DNA
• Protects the transcript from degredation / translation

Eukaryotic Genes

• Eukaryotic genes are split into exons and introns
• Exons are regions of DNA within a gene unit found in mature mRNA and are = “expressed”
• Introns are regions of DNA within a gene unit that are not found in mature mRNA and are = “intervening” regions

Splicing

• The spliceosome removes introns
• Some pre-mRNAs can be spliced differently, yielding different protein products from the same gene
• Splicing and capping of eukaryotic mRNAs often happen while transcription is ongoing, is co-transcriptional

Part C Summary

• Eukaryotic mRNAs need processing before export
• The unprocessed transcript is pre-mRNA or hnRNA
• Eukaryotic genes split into exons and introns
• Exons are in mRNA/expressed, Introns spliced, capped/polyadenylated whilst ongoing

Eukaryotes vs prokaryotes quiz

• Eukaryotic mRNA is not polyadenylated at the 3' end, it needs export
• In eukaryotes, mature mRNA has 7-methylguanosine attached by a 5' to 5' triphosphate bridge
• RNA polymerase II has a C-terminal domain tail which recruits capping, splicing and polyadenylation factors.
• Splicing is the removal of introns from pre-mRNA, not mature mRNA
• Transcription occurs in prokaryotes cytoplasm and eukaryotes nucleus