RNA Processing and Gene Expression
RNA Molecules and RNA Processing
Prokaryotes vs Eukaryotes
In prokaryotes:
Transcription and translation occur in the same compartment and simultaneously.
As the 3' end of RNA is undergoing transcription, the 5' end is being translated.
In eukaryotes:
Transcription occurs in the nucleus.
Translation occurs in the cytoplasm.
Experiment and Observations
Methods
Mix DNA with complementary RNA and heat to separate DNA strands.
Cool the mixture:
Complementary sequences pair.
DNA may reanneal with its complementary strand or with RNA.
Results
Noncoding regions of DNA are observed as loops.
Gene Structures – Exons & Introns
Cytochrome b gene and Ovalbumin gene consist of exons (coding) and introns (noncoding).
DNA Transcription: Conducted in the following stages:
DNA is transcribed into RNA.
Introns are removed by RNA splicing.
Example of Gene Sizes
Table 14.8: Contrasting Human Gene Size, mRNA Size, and Number of Introns
Gene | Gene Size (kb) | mRNA Size (kb) | Number of Introns
Insulin | 1.7 | 0.4 | 2
Rabbit ẞ-globin | 38.0 | 5.0 | 50
Collagen | 1150 | 246 | 576
Albumin | 25.0 | 2.1 | 14
Phenylalanine hydroxylase | 90.0 | 2.4 | 12
Dystrophin | 2000.0 | 17.0 | 50
Chicken ovalbumin | | |
mRNAs - Prokaryotes vs Eukaryotes
Prokaryotic mRNA:
Polycistronic (coding sequences can be followed by multiple genes).
No 5' cap or poly(A) tail.
Eukaryotic mRNA:
Monocistronic (only one protein coding sequence per mRNA).
Includes a 5' cap and a poly(A) tail (AAAAA).
mRNA Processing in Eukaryotic Systems
Table 14.2: Posttranscriptional Modifications to Eukaryotic pre-mRNA
Modification | Function
Addition of 5' cap | Facilitates ribosome binding, increases mRNA stability, enhances RNA splicing.
3' cleavage and addition of poly(A) tail | Increases stability, facilitates ribosome binding to mRNA.
RNA splicing | Removes introns, facilitates export of mRNA to cytoplasm.
RNA editing | Alters nucleotide sequence of mRNA.
Primary Transcript to Mature mRNA
Process Overview:
Transcription of prerequisite genes leads to the formation of a primary transcript.
5' capping, splicing, and polyadenylation form the mature mRNA.
The 5' cap is added shortly after ~25 nucleotides of mRNA are synthesized.
5' Capping
Steps:
Phosphatase removes phosphate group.
Guanyl transferase adds GMP (GTP) via a 5' to 5' guanylyl linkage.
Methyl transferase adds a methyl group to the guanosine.
Splicing Mechanism
Splicing occurs via the spliceosome, which consists of small nuclear RNAs (snRNAs):
Spliceosome components include:
U1, U2, U4, U5, and U6 snRNPs
Key steps include forming a lariat structure from the intron and cleaving at splice sites to join exons.
3' Cleavage and Polyadenylation
Mechanism described:
Pre-mRNA cleavage occurs downstream of the consensus sequence (specific sequences like AAUAAA).
Addition of adenine nucleotides (polyadenylation) generates the poly(A) tail at the 3' end.
Alternative Splicing
Provides multiple mRNA isoforms from a single gene through:
Removal of different introns/exons to yield various mature mRNA products.
Example: α-tropomyosin Gene
Different isoforms (striated muscle mRNA, smooth muscle mRNA, brain mRNA) generated by alternative splicing.
Tissue Specific Isoforms
Different cellular contexts lead to variable processing:
In thyroid cells, certain exons are included or excluded during alternative splicing compared to brain cells.
rRNA Splicing
Group I and Group II Introns
Group I introns can self-splice; Group II introns form lariat structures.
Ribozymes: RNA molecules that catalyze their own splicing; examples in mitochondrial and chloroplast rRNA genes.
Major Types of Introns
Group I: Mitochondrial and chloroplast rRNA (self-splicing).
Group II: Protein-coding genes (self-splicing).
Protein-encoding genes in the nucleus are