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, mRNA-like 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

2. Positioning of A nucleotide for transesterification reaction

#1

3. Positioning of 3’ splice site near exon1 for transesterification reaction #2

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.

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 determinedFirst disease related gene to be cloned

Model for the structure, regulation, and function of human genes

Tetramer2 alpha-like globins2 beta-like globins4 heme groups (1/globin)Require equal amounts of alpha and beta globins for proper Hbconcentration in RBCs

Mutations in beta globin splice sites reduce amount of hemoglobin resulting in beta thalassemia

Structure and Function of Hemoglobin

Mutations in beta thalassemia

Mutations in splice sites- reduce usage of proper splice site- no splicing or use of a cryptic splice siteMutations in introns or exons- creates new splice site- competes with proper site- reduces usage of proper splice site

Splicing mutations causing beta thalassemia