Gene-Proteins-MutationsA tutorial exploring how genetic mutations

affect proteins structure and function

Before you begin…

• Read the opening case study (Meeting the Affected Family) on the first page of lab - Discovering the Genetics and Molecular Biology of Sickle Cell Anemia .

As you explore this tutorial, think about the question posed in Part I of the Sickle Cell Anemia-Malaria lab case study:

Meeting the Affected Family:

Why do you think that Dr. Dufall wants a blood sample from Emily and Chaka, if neither of them has sickle cell anemia? In other words, what could looking at their hemoglobin tell us about this genetic condition?

Genes code for polypeptides!

One gene codes for the alpha globin polypeptides!

One gene codes for the beta globin polypeptides!

Remember that the hemoglobin protein is comprised of 4 polypeptide chains (2 alpha-globin chains and 2 beta-globin chains).

How do cells use the information encoded in genes to make proteins…

NucleusCytoplasm

Makes a mRNA copy (or transcript) of the

gene.

The ends of the mRNA transcript are modified

to facilitate transport from the nucleus.

Regions of the mRNA transcript that don’t

provide instructions for making a polypeptide

are removed (introns). The remaining mRNA

sequences (exons) are spliced back together.

mRNA leaves the nucleus and combines with a ribosome in the cytoplasm.

The ribosome “reads” the mRNA sequence and use the information to build a polypeptide (a chain of amino acids).

Transcription…in a little more detail!

First, watch an animation of transcription (click on picture above) to get a sense of how the whole process proceeds. Return to this show and continue after viewing the video.

Let’s first examine the structure of RNA more closely…

DNA RNA

Double stranded helix

Single stranded

Nucleotides are: C,G,A,T

Ribonucleotides are: C,G,A,U

A DNA nucleotide:

A RNA ribonucleotide:

DNA RNA

During transcription of mRNA notice that:

• Adenine (DNA) pairs with Uracil (RNA)

• Guanine (DNA) pairs with Cytosine (RNA)

• Cytosine (DNA) pairs with Guanine (RNA)

• Thymine (DNA) pairs with Adenine (RNA)

DNA

mRNA

Initiating transcription…

RNA polymerase begins to pry open the DNA and transcribes the gene using one of the strands as a template. Other transcription factors facilitate the process and regulate the expression of the gene.

New ribonucleotides are added according to nitrogenous base pairing rules (U=A; G=C).

DNA

mRNA Processing

These complexes (called splicosomes) chop out whole sections of mRNA called intervening sequences (aka introns) and splices together the expressed sequences (aka exons).

mRNA

An interesting process happens during processing. A complex of proteins and a kind of RNA (snRNA) grabs the mRNA forming loops…like this!

From The Cartoon Guide to Genetics by Larry Gonick

Spliceosomes…

Sequences of snRNA associated with proteins that recognize introns by binding to specific sequences of RNA, “chop” out the intron and splice the exons together!

mRNA processing…

Introns – intervening sequences that are not expressed.

Exons – expressed sequences..these are translated into a polypeptide.“5’ Caps” and “Poly-

A Tails” are ribonucleotides that are added to facilitate export of mRNA from the nucleus and to prevent the degradation of mRNA in the cytoplasm.

Making polypeptides from the instructions now encoded in mRNA!

Translating the genetic code!

Click on the picture above to watch an animation of the process of protein translation to get a sense of how this process occurs.

Remember that polypeptides are composed of specific sequences of amino acids!

The DNA sequence for a gene determines the amino acid sequence.

An example: The insulin polypeptide!

A hormone that regulate blood sugar levels in humans!

Each circle represents and amino acid whose name is abbreviated with 3 letters. “Ala” in this case stands for the amino acid Alanine.

Here are the 20 amino acids that comprise all polypeptides!

Notice that it is the “R-groups” (highlighted in white) that differ between each amino acid.

Also notice that amino acids have different chemical characteristics (non-polar, polar, and electrically charged (- and +))

- charge + charge

• The mRNA transcript is read three “letters” (aka ribonucleotides) at a time.

• The triplet is called a codon.

• Codons act as the code that tells the ribosome the order in which to link amino acids during translation.

The genetic code!

Table of mRNA codons and the amino acids for which they code!

Notice that the codons are somewhat “redundant”. That is, several different codons can code for the same amino acid.

Also notice that 3 codons serve as “STOP” codons. These signal the end of the gene!

A mutation can alter the DNA sequence for a gene which can

cause a change in the mRNA sequence which can result in a

change in the amino acid sequence of the polypeptide.

This can alter the structure and therefore function of the protein!

Types of mutations…Normal mRNA from the gene

A base-pair substitution, which replaces one DNA nucleotide for another can have different effects on the polypeptide depending on the nature of the mutation.

Just so you know…

These changes occurred as mutations in the DNA, and resulted in a change in the mRNA, depicted in the figure.

Types of mutations…Frameshift mutations…shift the reading frame during translation resulting in many changes in the amino acid sequence downstream of the mutation!

Normal mRNA from the gene

Thought Question…

What effect do you think each of the mutations discussed so far would have on the structure (size & shape) and function of the polypeptide?

Be sure to complete the homework for the Discovering the Genetics and Molecular Biology lab in your lab manual prior to coming to lab.

Back to the case studyMeeting the Affected Family:

Why do you think that Dr. Dufall wants a blood sample from Emily and Chaka, if neither of them has sickle cell anemia? In other words, what could looking at their hemoglobin tell us about this genetic condition?