BIO 222 Chap 14: Eukaryotic Gene Structure

Gene Organization

• Bacterial genes are colinear with proteins, but eukaryotic genes often are not.
• The concept of colinearity suggests that a continuous sequence of nucleotides in DNA encodes a continuous sequence of amino acids in a protein.
• A continuous sequence of nucleotides in DNA codes for a continuous sequence of amino acids in a protein, therefore the number of nucleotides in the gene is proportional to the number of amino acids in the protein.
• The noncolinearity of eukaryotic genes was discovered by hybridizing DNA with the mRNA transcribed from it.
• When DNA was hybridized to the mRNA transcribed from it, regions of DNA that did not correspond to RNA looped out, showing evidence that eukaryotic genes are not colinear with the proteins they encode.

Introns

• Many eukaryotic genes contain exons and introns
• Both exons and introns are transcribed into RNA, but introns are later removed by RNA processing.
• The numbers and sizes of introns vary from gene to gene.
• Introns are common in eukaryotic genes but uncommon in bacterial genes.
• The coding sequences of many eukaryotic genes are disrupted by noncoding introns.

Major Types of Introns

• Group I introns are located in the genes of bacteria, bacteriophages, and eukaryotes and splice through self-splicing.
• Group II introns are located in the genes of bacteria, archaea, and eukaryotic organelles and splice through self-splicing.
• Nuclear pre-mRNA are located in protein-encoding genes in the nucleus of eukaryotes and splice through spliceosomal mechanisms.
• tRNA are located in tRNA genes of bacteria, archaea, and eukaryotes and splice through enzymatic mechanisms.

The Concept of a Gene

• Genes include DNA sequences that code for all exons and introns
• Includes those sequences at the beginning and end of the RNA that are not translated into a protein, including the entire transcription unit
  • The promoter
  • The RNA coding sequence
  • The terminator

Messenger RNA Structure

• A mature mRNA contains a 5' untranslated region (5' UTR, or leader sequence)
  • Shine-Dalgarno sequence
  • Protein-coding region
  • 3' untranslated region
    • The 5' and 3' untranslated regions do not encode any amino acids of a protein, but contain information that is important for RNA stability and regulation of translation
• Three primary regions of mature mRNA are the 5' untranslated region, the protein-coding region, and the 3' untranslated region.
• Brenner, Jacob, and Meselson demonstrated that ribosomes do not carry genetic information.

Pre-mRNA Processing

• Pre-mRNA processing includes the addition of the 5' cap.
  • A nucleotide with 7-methylguanine; 5'-5' bond is attached to the 5' end of the RNA
• Pre-mRNA processing includes the addition of the poly(A) tail.
  • ~ 50-250 adenine nucleotides are added to the 3' end of the mRNA

Posttranscriptional Modifications To Eukaryotic Pre-mRNA

• The addition of the 5' cap facilitates binding of ribosome to 5' end of mRNA and increases mRNA.
• 3' cleavage and addition of poly(A) tail increases stability of mRNA and facilitates binding of ribosome to mRNA.
• RNA splicing removes noncoding introns from pre-mRNA, facilitates export of mRNA to cytoplasm, and allows for multiple proteins to be produced through alternative splicing
• RNA editing alters nucleotide sequence of mRNA
• Most eukaryotic mRNAs have a 5' cap that consists of a nucleotide with 7-methylguanine attached to the pre-mRNA by a unique 5'-5' bond
• Most eukaryotic mRNAs have a 3' poly(A) tail.
• A protein that adds the 5' cap is associated with RNA polymerase II, which transcribes pre-mRNAs, but is absent from RNA polymerases I and III, which transcribe rRNAs and tRNAs, respectively.

RNA Splicing

• RNA Splicing requires consensus sequences
  • 5' consensus sequence: GU A/G AGU: 5' splice site
  • 3' consensus sequence: CAGG
  • Branch point: the adenine “A”: ~18-40 nucleotides upstream of 3'-splicing site
• Spliceosome: five RNA molecules + 300 proteins
• Splicing of nuclear pre-mRNA introns is a two-step process:
  1. The 5' end of the intron is cleaved from the upstream exon and attached to the branch point to form a lariat.
  2. The 3' end of the intron is cleaved from the downstream exon, and the ends of the two exons are spliced together.
• In the process, the intervening intron is removed.
• These reactions take place within the spliceosome.
• If a splice site were mutated so that splicing did not take place, the protein encoded by the mRNA would be longer than normal.
• RNA splicing takes place within the spliceosome and assembles sequentially.

Nuclear Organization

• Intron removal, mRNA processing, and transcription take place at the same site in the nucleus
• Minor splicing
  • Two types of self-splicing introns: group I introns and group II introns.
  • These introns have complex secondary structures that enable them to catalyze their own excision from RNA molecules without the aid of enzymes or other proteins
• Group I and group II introns fold into characteristic secondary structures.
• Self-splicing introns are in some rRNA genes in protists and in mitochondria genes in fungi
• Alternative processing pathways for processing pre-mRNA
  • Alternative splicing enables exons to be spliced together in different combinations to yield mRNAs that encode different proteins
  • Multiple 3' cleavage sites allow pre-mRNA to be cleaved and polyadenylated at different sites
• Alternative 3' cleavage sites result in multiple mRNAs of different lengths.
• Pre-mRNA encoded by the gene for calcitonin undergoes alternative processing.

RNA Editing

• RNA editing: coding sequence altered after transcription.
• Guide RNAs play a role.
• Trypanosoma brucei causes African sleeping sickness with mRNA produced from mitochondrial genes undergoing extensive RNA editing
• Guide RNA adds nucleotides to the mRNA that were not encoded by the DNA.

Transfer RNA Structure

• Rare modified RNA nucleotide bases
  • Ribothymine
  • Pseudouridine
• Common secondary structure-the cloverleaf structure
• Anticodon
• Two of the modified bases found in tRNAs are produced by the chemical alteration of the four standard RNA bases.
• Rare bases are incorporated into tRNAs by chemical changes in one of the standard bases
• All tRNAs possess a common secondary structure, the cloverleaf structure.
• Transfer RNAs are processed in both bacterial and eukaryotic cells

Ribosomal RNA

• Large ribosome subunit
• Small ribosome subunit
• Changes that take place in rRNA processing:
  • Methylation of bases
  • Cleavage of bases
  • Nucleotides trimmed from the ends of rRNAs

Ribosomes

• Bacterial (70S) ribosome subunits:
  • Large (50S) = 23S (2900 nucleotides), 5S (120 nucleotides)
  • Small (30S) = 16S (1500 nucleotides)
• Eukaryotic (80S) ribosome subunits:
  • Large (60S) = 28S (4700 nucleotides), 5.8S (160 nucleotides), 5S (120 nucleotides)
  • Small (40S) = 18S (1900 nucleotides)

Numbers of rRNA Genes In Different Organisms

• Escherichia coli = 7
• Yeast = 100-200
• Human = 280
• Frog = 450
• Long noncoding RNAs (lncRNAs) are long RNA molecules that do not encode proteins
• Many of these molecules function in the control of gene expression
• Enhancer RNAs (eRNAs) are transcribed from enhancers and also play a role in control of gene expression
• Circular noncoding RNAs (circRNAs) may serve as decoys for miRNAs
• In prokaryotic cells CRISPR RNAs function in defense against foreign DNA

Small RNA Molecules

• RNA interference: limits the invasion of foreign genes and censors the expression of their own genes
• Types of small RNAs
• Processing and function of microRNAs
• Small interfering RNAs (siRNAs) and microRNAs (miRNAs) are produced from double-stranded RNAs
• An siRNA or miRNA combines with proteins to form a RISC, which then pairs with mRNA through complementary pairing between bases on the siRNA or miRNA and bases on the mRNA.
• Piwi-interacting RNAs (piRNAs) are primarily found in the germ cells of animals and inhibit transposons

Differences Between SiRNAs and MicroRNAs

• SiRNAs originate from mRNA, transposons, or viruses while miRNAs originate from RNA transcribed from distinct genes.
• SiRNAs cleave of RNA duplex or single-stranded RNA that forms long hairpins while miRNAs cleave single-stranded RNA that forms short hairpins of double-stranded RNA
• Both siRNAs and miRNAs are 21-25 nucleotides in Size.
• SiRNAs cause degradation of mRNA, inhibition of transcription, and chromatin modification while miRNAs cause degradation of mRNA, inhibition of translation, and chromatin modification.
• SiRNAs’ target: Genes from which they were transcribed
• miRNAs’ target: Genes other than those from which they were transcribed