RNA Structure and Synthesis - Eukaryotic Transcription

RNA Structure and Synthesis (II) - Transcription of Eukaryotic Genes

• Eukaryotic cells have three distinct RNA polymerase classes, each recognizing specific gene types.
• RNA polymerase I synthesizes the precursor for large ribosomal RNAs (28S, 18S, and 5.8S) within the nucleolus.
• RNA polymerase II synthesizes mRNA precursors and some small nuclear RNAs (snRNAs).
• Promoters (class II genes) are specific DNA sequences within the eukaryotic promoter region.
• These sequences are recognized by RNA polymerase II.
• Sequences include the CAAT box, and the Hogness (TATA) box
• Length ranges from approximately 45 to 55 bases.
• Many promoters also contain a GC box (GGGCGG) sequence. These act as cis-acting genetic elements – meaning they're on the same DNA molecule as the transcribed gene.
• These sequences serve as binding sites for general transcription factors.
• Eukaryotic genes have enhancer sequences which are also cis-acting genetic elements.
• These elements increase transcription initiation rates by RNA polymerase II.
• Enhancers need to be on the same chromosome as the target gene.
• They can be located upstream or downstream of the transcription start site. They can be relatively close to or thousands of bases distant from the promoter.
• They can be located on either strand of DNA.
• Enhancers stimulate RNA polymerase II by bending the DNA, allowing interaction with the transcription initiation complex.
• Enhancers contain DNA sequences called response elements that bind specific transcription factors called activators.
• RNA polymerase II is inhibited by α-amanitin, a potent toxin from the mushroom Amanita phalloides.
• α-amanitin forms a tight complex with RNA polymerase II, therefore inhibiting mRNA synthesis and protein synthesis.
• Mitochondria contain a single RNA polymerase that closely resembles bacterial RNA polymerase rather than a typical eukaryotic enzyme.
• Ribosomal RNA (rRNA) of both prokaryotes and eukaryotes originate from long precursor molecules called preribosomal RNA.
• In eukaryotes, the 28S, 18S and 5.8S rRNAs are produced from a single precursor molecule.
• In prokaryotes, 23S, 16S, and 5S rRNAs are produced from a single precursor.
• Eukaryotic 5S rRNA is synthesized by RNA polymerase III and subsequently modified.

Post-transcriptional Modification of RNA

• Transfer RNA (tRNA)

• Mature tRNA contains a 16-nucleotide sequence at the 5' end, and a 14-nucleotide intron contained in the anticodon loop area.

• Uracil residues at the end are replaced by CCA sequences.

• Messenger RNA (mRNA)

• Synthesized by RNA polymerase II

• Eukaryotic mRNAs have modifications:

• 5' capping

• Addition of a poly-A tail

• Removal of introns

• Alternative splicing

5' Capping

• The 5' cap is a 7-methylguanosine attached backward to the 5' terminal end.
• This creates an unusual 5'→5' triphosphate linkage.
• Adding the guanosine (guanylyltransferase) is a nuclear process.
• Methylating the terminal guanine is a cytoplasmic process (guanine-7-methyltransferase) using S-adenosylmethionine as a methyl group donor.

Addition of a Poly-A Tail

• A chain of 40-200 adenine nucleotides is added to the 3' end.
• This poly-A tail is not part of the initial DNA sequence but is added after transcription by an enzyme (polyadenine polymerase).

Polyadenylation Signal Sequence

• AAUAAA sequence found near the 3' end of the RNA molecule signals the addition of the poly-A tail.
• Tails help stabilize mRNAs and promote their export from the nucleus after synthesis.
• The tail is gradually degraded after mRNA exits the nucleus and enters the cytosol.

Removal of Introns

• Eukaryotic mRNA maturation involves removing non-protein-coding RNA sequences called introns from the primary transcript.
• Exons (coding sequences) are then spliced together to form the mature mRNA.
• The molecular machine that performs this splicing is called a spliceosome.

Alternative Splicing of mRNA Molecules

• Pre-mRNA from some genes can be spliced several ways
• Gives rise to multiple mRNA forms (and therefore proteins).
• A method for producing a wide range of proteins from a limited number of genes.