Protein Synthesis
Laboratory Investigation

Objectives

Upon completion of this lab, you should be able to:

• understand the role played by the ribosomes and the three RNA types
• understand the meaning of the terms transcription and translation
• develop an RNA transcript from the DNA template
• match the appropriate codon-anticodon sites to produce an appropriate polypeptide

Introduction

Protein synthesis occurs in the cytoplasm of eukaryotes, along the surface of the ribosomes. A eukaryotic cell may have thousands of ribosomes, and each has two parts or subunits. The large and small subunit are both made of rRNA plus protein. A cluster of ribosomes on the same mRNA is called a polyribosome or polysome. Use the following link to learn more about the ribosomes and their role in protein synthesis.

Ribosome Structure

Since DNA remains in the nucleus, another molecule must carry the instructions of the DNA to the site of protein synthesis. This other molecule is RNA, or ribonucleic acid. RNA consists of a single linear strand of nucleotides that can twist and fold into a variety of shapes. Each nucleotide consists of a ribose sugar, a phosphate group, and one of four bases, A, C, G, and Uracil (U) instead of thymine (T). Thus, RNA is formed using a portion of DNA as a template.

Use the following animation, Overview of Protein Synthesis, to review the basics of protein synthesis. Go to DNA Workshop Activity and watch the Protein Synthesis animation.

The Process – Transcription

The process of protein synthesis starts with a portion of the DNA molecule being used as a template for production of mRNA. This process is called transcription. During transcription, a portion of DNA unwinds and serves as a template to produce an RNA transcript. Each region of DNA can be transcribed thousands of times during the life of a cell. View the following animation to watch the process of transcrition.

The next segment of protein synthesis requires that mRNA and tRNA line up to position amino acids in the appropriate positions, creating the polypeptide. This is called translation. During translation, three types of RNA convert the message of DNA into the sequence of amino acids that is the primary structure of the protein.

1. Ribosomal RNA (rRNA) combines with proteins to form the ribosomes
2. Messenger RNA (mRNA) carries the "blueprint" to the ribosome.
3. Transfer RNA (tRNA) brings the correct amino acid to the ribosomal position of messenger RNA required for protein synthesis.

The Process – Translation

Nucleic acids must construct “code words” from four kinds of nucleotides to designate each of the twenty amino acids found in a polypeptide chain. A sequence of three nucleotides (a triplet) provides 64 choices (43), more than enough to specify the twenty amino acids typically used to construct proteins in eukaryotic cells. Crick, Brenner, and others deduced that the nucleotide bases are read three at a time and that a “start” signal establishes the correct “reading frame.”

The genetic code consists of 61 triplets that specify amino acids and 3 that serve to stop protein synthesis (see the table below). Each triplet of nitrogen bases that codes for an amino acid is called a CODON. The code is universal for all life forms, with few exceptions.

Codon-Anticodon Interaction

1. Each kind of tRNA has an anticodon triplet that is complementary to a mRNA codon.
2. After the mRNA arrives in the cytoplasm, an anticodon on a tRNA bonds to the codon on the mRNA. This should properly align a correct amino acid into position.
3. Often, only the first two bases of an anticodon must be precisely complementary; the third base may vary (the "wobble" effect).

Stages of Translation

1. complex of ribosomal units, mRNA, and tRNA forms.
2. Transfer RNAs bring appropriate amino acids to the site.
3. An enzyme forms a peptide bond between the newly arrived amino acid and the growing polypeptide chain.
4. The bond between the old amino acid and its tRNA is broken, and the old tRNA leaves the complex.
5. Once a stop codon is reached, the polypeptide chain detaches.
6. Enzymes called release factors are involved in the detachment process.
7. The polypeptide can join the cytoplasmic pool of proteins or be further processed by the cytomembrane system.

Click here for an animation showing translation.

Overview of the process of Protein Synthesis

A fun activity showing the process of protein synthesis.

		U	C	A	G
	U	phenyl-alanine	serine	tyrosine	cysteine	U
						C
		leucine		stop	stop	A
				stop	tryptophan	G
	C	leucine	proline	histidine	arginine	U
						C
				glutamine		A
						G
	A	isoleucine	threonine	asparagine	serine	U
						C
		*methionine		lysine	arginine	A
						G
	G	valine	alanine	aspartic acid	glycine	U
						C
				glutamic acid		A
						G

Information about the genetic table.

Question 1

The following are the mRNA sequences for normal and sickle-cell hemoglobin.

Normal: 5’ AUG GUG CAC CUG ACU CCU GAG GAG AAG UCU GCC 3’
Sickle-cell: 5’ AUG GUG CAC CUG ACU CCU GUG GAG AAG UCU GCC 3’

a) Determine the template DNA code for each.
b) Determine the amino acid sequence of each.

You will need to do a little research to answer the next two questions.

c) Why does this single amino acid substitution cause the terrible symptoms of sickle-cell disease? What characteristics of the amino acids involved in this substitution cause the cell to take on their deformed shapes?
d) How can these differences in hemoglobin be detected in the lab?

Question 2

The DNA sequence below represents the template strand of a gene that codes for GOOP, the polypeptide that gives Spider-Man his unique ability to stick to walls. Researchers around the world have been trying for years to find the elusive gene that codes for the mutated version of GOOP (Spider-Man’s version), which has been shown to undergo a conformational change (a change in shape) in response to unforeseen peril. The goal of the project is to genetically engineer police dogs that are able to climb up walls to prevent criminals from getting away. Since animals can often sense danger from catastrophes such as earthquakes, genetically engineered mice with this gene would be able to sense the danger and start to climb up walls, thus giving a warning prior to a number of catastrophes.

5’ tac agc ttc agt tca agt tgt gtc gcc ttt caa tgt ttt acc att aag gat caa ttt gta agt agt cat 3’

a) What is the resulting mRNA sequence (5’ to 3’)?
b) What are the first four and the last four amino acids in GOOP?

Researchers were able to obtain a sample of Spider-Man’s DNA from a spot of blood left behind after his last encounter with the Green Goblin. They determined that Spider-Man has two versions of GOOP that both differ from that of normal (wild-type) by one nucleotide. The researchers hypothesize that the version that deviates the most from that of wild-type is most likely the version that gives Spider-Man his mutant ability.

c) Which of the two versions of GOOP is most likely to yield a protein that differs from that of wild type? Why (answer in one sentence)?

GOOP Version 1
5’ tac agc ttc agt tca agt tgt gtc gcc ttt caa tgt ttt acc att aac gat caa ttt gta agt agt cat 3’ (DNA template strand)

GOOP Version 2
5’ tac agc ttc agt tca agt tgt gtc gcc ttt caa tgt ttt acc ata aag gat caa ttt gta agt agt cat 3’ (DNA template strand)