Lab VI - Beta Globin

Lab VI: Beta-Globin Gene What is happening today? In lab today, you will examine DNA and protein sequence data for the beta-globin gene and by so doing discover how that information is expressed as beta-globin mRNA and polypeptide . Background Beta-globin comprises two of the four subunits in hemoglobin (Fig 1). Stated in its simplest terms, the expression of a coding gene involves the major steps known as transcription and translation. These steps are part of the larger foundational concept in molecular biology known as the Central Dogma. Only one strand of dsDNA encodes a gene, hence the direction in which the information is encoded by the template strand depends on whether the W or C strand reflects the “ sense ” of the transcript (Fig 3). Figure 1. Beta-globin subunits in this Figure 2. Central dogma of molecular biology.

diagram of hemoglobin are indicated. Note information flow in gene expression. Figure 3. Arrows along the sense strands indicate the directionality of transcribed RNA.

• Where does it seem to prematurely end? RF: _ c __ Approximate Nucleotide Number: _ 62280 _______ In eukaryotes, a pre-mRNA transcript is LONGER than the protein coding sequence it encodes, since it includes sequences important to initiating and terminating translation (5’ and 3’ UTRs), and assisting with post-transcriptional events (introns). • Find the next exon (and any others needed to finish the protein sequence). RF of Exon 2: _ a __ Beginning Nucleotide #:_ 62409 ______ Ending Nucleotide #:_ 62630 ______ RF of Exon 3? _ b __ Beginning Nucleotide #:_ 63482 ______ Ending Nucleotide #:_ 63605 ______ (and if needed...) RF of Exon 4? ___ Beginning Nucleotide #:_______ Ending Nucleotide #:_______ Introns have specific structural sequences within them, and so do the adjoining exons. • What are the names of the 3 key structural sequences? __ 5’ consensus sequence __ ___ branch point ___ ___ 3’ consensus sequence ___ • What are the first 2 bases (on the sense strand) of each intron you find? You can deduce the point at which one codon begins and ends in the exons (or are even split between exons) by comparing the DNA sequence to the codons for the known amino acid sequence of beta-globin. 5’ dinucleotide sequence of the 1st intron is _ GT ___ & the 3’ dinucleotide seq. is _ AG __ 5’ dinucleotide sequence of the 2nd intron is _ GT ___ & the 3’ dinucleotide seq. is _ AG _ • What is the stop codon following the open reading frame for beta-globin? _ UGA ____ • Polyadenylation signals in the 3 ’-UTR identify the location at which the transcript is cleaved and a poly-A tail is added. The polyadenylation sequence AAUAAA is highly conserved among mammals. Find it. Show to instructor when you find it. _KK___ ☚ Instructor’s initials. What is the beginning nucleotide # of the sequence? _ 63718 __ Hemoglobin Model • Examine the hemoglobin model with the instructor before you leave the lab. Sickle cell is due to a mutation in beta-globin polypeptides. Using the old nomenclature,

hemoglobin S is a defective tetramer containing two mutant beta- globins and two wild- type alpha-globins. The mutation in the beta-globin gene is referred to as HbS and oddly the wild-type beta-globin is referred to as HbA (this nomenclature predates our current understanding of the protein). • Sickle cell is due to the mutation of a single nucleotide in the beta-globin gene that changes the seventh codon of beta-gl obin mRNA so that instead of a glutamate a valine is used to build the protein during translation. In mature HbA and HbS it is the sixth amino acid that is altered as the first amino acid is removed post-translationally. What enzyme performs this function? _ glutamic acid _____________ Clean up. Wipe-down your scroll with a damp paper towel to remove any ink you marked it with. Dry it as you go with a dry paper towel. Roll it back up to the beginning of the gene. RETURN SCROLL (with rubberband around it) & PEN TO INSTRUCTOR. Post-lab Questions: To be handed-in at the beginning of next week’s lab. Submit the answers to the following questions, along with the in-class activity, by Friday evening. Q1. Does the ribosome switch reading frames as it translates the gene? The ribosome's function is to maintain the mRNA sequence being translated in the correct reading frame. However, certain viral mRNAs contain unique sequence information and structural components that lead to ribosome slippage and a subsequent readjustment of the reading frame. Q2. How are the introns spliced out of the pre-mRNA? What are the recognition sequences for an intron? For an animation that illustrates the process, go to http://www.sumanasinc.com/webcontent/animations/content/mRNAsplicing.html . The cell receives questions on what to delete from a few brief nucleotide sequences. An RNA protein complex (snRNP) then displaces the proteins forming base pairs with the branch site after a branch point binding protein (BBP) and a helper protein (U2AF) first recognize the branch point site inside the intron. This snRNP is a component of a complex molecular assembly known as a spliceosome that comes together at the intron.