Download reduce usage of proper splice site

Lecture 4 Topic 2
Gene Function & Gene Expression
Topic 2 Material and Chapter 3
• Not Covering
– Transcription
• Transcription Termination in Bacteria
• Already covered:
– Gene Structure
• component parts of gene, template and non-template strand, mRNAlike strand, 5’ and 3’ end of gene
• Will cover in detail later
– Transcription
• Initiation of transcription, promoter structure, RNA polymerase
structure and function
• Will cover now
– steps in transcription, details of eukaryotic transcription
termination, RNA processing, translation, protein structure and
function as it relates to gene function and mutant phenotypes
Transcription Termination and mRNA
5’-End and 3’-End-Formation
Steps of Transcription
RNA processing during
transcription
- 7mG capping 5’ end
- 3’ end formation
5’ CAP
3’ Poly A Tail
RNA Splicing
• RNA splicing reactions
– Two transesterification reactions
• first cleaves at 5’ end of intron (5’ splice site)
• second cleaves at 3’ end of intron (3’ splice site)
• Self-Splicing RNAs (discovery of ribozymes)
– Group I and Group II introns remove themselves
• difference is in the first transesterification (see later)
• Group I introns use a free G nucleotide to catalyze reaction
• Group II splicing is similar reaction to that in pre-mRNA splicing
• pre-mRNA Splicing
– catalyzed by “spliceosome”
• multiple complexes of proteins and RNA (ribonucleoprotein particles
called snRNPs
– snRNAs called U RNAs (U1,2,4,5 and 6)
– splicing proteins
• snRNPs bind to pre-mRNA in a specific sequence to catalyze splicing
Transesterification Reactions
in RNA Splicing
• Attack of the sugar-phosphate bond at 5’
splice site
• Group 1 = 3’ OH of free G nucleotide
• Group 2 and pre-mRNA = 2’ OH group
of branch point A nucleotide
• Attack of the sugar phosphate bond at
the 3’ splice site
• 3’ OH of nucleotide at 3’ end of exon 1
• Intron released
• Group 1 introns = linear
• Group 2 and pre-mRNA introns = lariat
• Base pairing between U snRNAs and
intron sequences brings intron sequences
together and catalyzes the
transesterification reactions
Identification of Intron Sequences important for Splicing
U snRNA:pre-mRNA base pairing
1. Identification of 5’ splice site
and Branch Point
3. Positioning of 3’ splice site
near exon1 for
transesterification reaction #2
2. Positioning of A nucleotide
for transesterification reaction
#1
Summary of terms and processes
Mutations affecting Splicing
• Use mutation in globin genes to illustrate
effects of mutation on gene function
– splicing mutations
• mutations of splice sites
• mutation in exons creating cryptic splice site
– reduction in protein levels
– production of abnormal protein
– mutations in coding sequence
• reduction in protein levels
• production of abnormal proteins
Genetic Disorders of Hemoglobin
Hemoglobinopathies
hemoglobinopathy
disease, hematology> Disorder due to abnormalities in the hemoglobin
molecule, the best known being sickle cell anemia in which there is a
single amino acid substitution (valine for glutamate) in position 6 of the
beta chain. In other cases one of the globin chains is synthesised at a
slower rate, despite being normal in structure.
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Hemolytic anemias (RBC lysis)
Thalassemias (decreased amount to to unbalanced synthesis of one globins)
Methemoglobinemias (reduced oxygen transport due to defective hemoglobin)
Genetic Disorders of Hemoglobin
Hemoglobinopathies
Clinical Importance:
5 % world population carry alleles causing hemoglobinopathies!
Model for determining the molecular basis of disease:
One of first protein structures determined
First disease related gene to be cloned
Model for the structure, regulation, and function of human genes
Structure and Function of Hemoglobin
Tetramer
2 alpha-like globins
2 beta-like globins
4 heme groups (1/globin)
Require equal amounts of alpha
and beta globins for proper Hb
concentration in RBCs
Mutations in beta globin splice
sites reduce amount of
hemoglobin resulting in beta
thalassemia
Mutations in beta thalassemia
Mutations in splice sites
- reduce usage of proper splice site
- no splicing or use of a cryptic splice site
Mutations in introns or exons
- creates new splice site
- competes with proper site
- reduces usage of proper splice site
Splicing mutations
causing beta thalassemia