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Lab 1 2
NUCLEIC CIDS
OBJECTIVES
- (^) Identify the components of deoxyribonucleotid s and ribonucleotides;
- (^) Distinguish between DNA and RNA according o their structure and function;
- (^) Describe DNA replication, transcription, and tr nslation;
- (^) Give the base sequence of DNA or RNA when presented with the complementary strand;
- (^) Identify a codon and anticodon on RNA model and describe the location and function of each;
- (^) Give the base sequence of an anticodon when presented with that of a codon, and vice versa;
- (^) Describe what is meant by the one-gene, one- olypeptide hypothesis.
INTRODUCTION
By 1900, Gregor Mendel had demonstrated pa terns of inheritance, based solely on careful experimentation and observation. Mendel had no clear i ea how the traits he observed were passed from generation to generation, although the seeds of that kn wledge had been sown as early as 1869, when the physician-chemist Freid rich Miescher isolated the ch mical substance of the nucleus. Miescher found the substance to be an acid with a large phosphorus content and named it "nuclein." Subsequently, nuclein was identified as DNA, short for deoxyribonuc eic acid. Some 75 years would pass before th significance of DNA would be revealed. This exercise wil familiarize you with the basic structure of nucleic acids and their role in the cell. Understanding the unction of nucleic acids-both DNA and RNA (ribonucleic acid)-is central to understanding life itself.
MODELING THE STRUCTURE AND FUNCTION OF N CLEIC ACIDS
A Nucleic Acid Structure
Nucleic acids are long, chainlike molecules for ed by the linking together of smaller molecules called nucleotides. A nucleic acid, deoxyribonucleic cid or DNA, is the material which comprises the gene.
Procedure
- Obtain a DNA puzzle kit. It should contain th following components as shown in Figure 14-
- Select a single deoxyribose sugar, and ade ine base (labeled A), and a phosphate, fitting them together as shown in Figure 14-2. This is a single nucleotide (specifically a deoxyribonucleotide), a unit consisting of a sugar (deoxyribose), a phosphate group, and a nitr gen- containing base (adenine).
A
Figure 14-2 One deoxyribonucleotide.
A
(18) Deoxyibose (4) Adenine
N - Z H � N-H-- H CH3 O··
(6) Cytosine (4) Thymine
(9) Ribose
Figure 14-1 DNA pu zle kit components.
- Deoxyribose^ is a sugar compound contai^ ing five carbon atoms and one oxygen atom. Four of the five are joined by covalent bonds i to a ring (Figure 14-3). The three knobs represent covalent chemical bonds which deoxyribo e can form with the bases and phosphate. HO ' C H 2 HC
CH
OH
Figure 14-3 Structural for^ ula of deoxyribose.
There are four kinds of nitrogen-containin bases in DNA. Two are purines and are double- ring structures. Specifically, the two purines re adenine and guanine (abbreviated A and G, respectively); Figure 14-4.
Figure 14-2 One deoxyribonucleotide.
HO NH
........._CH20 OH H C -
HC I '--CH
Ribose HJI tH.d-J (^) -......_c
OH OH g
Figure 14-9 Ribose and the pyrimidine uracil.
- Compare the structural formulas of ribos^ and deoxyribose.
a. How do they differ?
b. Why is the sugar of DNA called a deo yribose.
B. Modeling DNA Replication
c-1/
NH (^) Uracil
DNA replication takes place during the S stag of interphase of the cell cycle. Recall that the DNA is aggregated into chromosomes. Before mitosis, the chromosomes duplicate themselves so that the daughter nuclei formed by mitosis will have the s me number of chromosomes (and hence the same amount of DNA) as did the parent cell.
Replication begins when hydrogen bonds bet een nitrogen bases break and the two DNA strands "unzip." Free nucleotides within the nucleus bo d to the exposed bases, thus creating two new strands of DNA. The process of replication is con rolled by enzymes call DNA polymerases.
Procedure
- Construct eight more deoxyribonucleotide (two of each kind) but don't link them into strands.
- Now return to the double-stranded DNA s gment you constructed earlier. Separate the two strands, imagining the zippier-like fashion n which this occurs within the nucleus.
- Link the free deoxyribonucleotides to each of the "old" strands. When you are finished, you should have two double stranded segmen s.
Note that one strand of each is the parental (" Id") strand and the other is newly synthesized from free nucleotides. This illustrates the semi-co servative nature of DNA replication. Each of the parent strands remains intact-it is conserved and a new complementary strand is formed on it. Two "half-old, half new" DNA molecules result.
D. Translation: RNA to Protein
Once in the cytoplasm, mRNA strands att ch to ribosomes, on which translation occurs. To translate means to change from one language t another. In the biological sense, translation is the conversion of the linear message encoded on mRNA to a linear strand of amino acids to form a polypeptide. (A peptide is two or more amino ac‚ds linked by a peptide bond.)
Translation is accomplished by the interacti n of mRNA, ribosomes, and transfer RNA (tRNA), another type of RNA. The tRNA molecule is for ed into a four-cornered loop. You can think of tRNA as a baggage-carrying molecule. Within the cyt plasm, tRNA attaches to specific free amino acids. This occurs with the aid of activating enzymes, represented in your model kit by the pieces labeled "glycine activating" or "alanine activating." The amino acid-carrying tRNA then positions itself on ribosomes where the amino acids become linke together to form polypeptides.
Procedure
- Obtain three tRNA pieces, three amino a id units and three activating units.
- Join the amino acids first to the activatin^ units and then to the tRNA.
a. Will a particular tRNA bond with any mino acid, or is each tRNA specific?
- In the space below, list the sequence of b ses on the mRNA strand, starting at the left (3' end).
3' (left end) 5' (right end)
Translation occurs when a three-base seq ence on mRNA is "read" by tRNA. This three- base sequence on mRNA is called a codon. Think f a codon as a three-letter word, read right (5') end to left (3') end.
a. What is the order of the rightmost (firs ) mRNA codon? (Remember to list the letters in the reverse order of that in the mRNA equence.)
The first codon on the mRNA model i (5' end)
3' (left end)
- Slide the mRNA strand onto the ribosome emplate sheet, with the first codon at the 5' end.
- Find the tRNA-amino acid complex that complements (will fit with) the first codon. (^) The complementary three-base sequence on t e tRNA is the anticodon. Binding between codons and anticodons begins at the P site of the 40s subunit (the smaller subunit) of the ribosome. The tRNA-amino acid complex with the co rect anticodon positions itself on the P site.
b. Record the tripeptide that you ave just modeled in the space below.
You have created a short p lypeptide. Polypeptides may be thousands of amino acids in len th. As you see, the amino acid sequence is ultimately determin d by DNA, because it was the original source of information.
- Finally, let's turn our attention t o t e concept of a gene. A gene is a unit of inheritance. Our current u derstanding of a gene is that a gene codes for one polypeptide. T is is appropriately called the one- gene, one-polypeptide hypothe is.
a. Given this concept, do you thi k a gene consists of one, several, or many deoxyribonucleo ides?
A gene probably consists o f _ - - -_ _ _ _ deoxyribonucleotides.
PLEASE DISASSEMBLE YOU MODELS AND RETURN
THEM T O T EIR RESPECTIVE COLOR-
CODED BO ES.
- Move the tRNA-amino acid complex onto the P site on the ribosome template sheet and fit the codon and anticodon together. In the boxE s below, indicate the codon, anticodon, and the specific amino acid attached to the tRNA.
mRNA codon1 =
tRNA anticodon1 =
amino acid1 =
- Now identify the second mRNA codon and fill in the boxes.
mRNA codon2 =
tRNA anticodon2 =
amino acid2 =
= mRNA codon
= tRNA anticodon
= amino acid
- The second tRNA-amino acid complex mo^1 es onto the A site of the 40s subunit of the ribosome. Position this complex on the A s te. An enzyme now catalyzes a condensation reaction, forming a peptide bond and linkin J the tow amino acids into a dipeptide. (Water, H2O. is released by this condensation reaction.)
- Separate amino acid1 from its tRNA and lin^ it to amino acid2. (In reality, separation occurs somewhat later, but the puzzle doesn't allow his to be shown accurately.)
mRNA codon2 =
tRNA anticodon2 =
amino acid2 =
! Enzymatic
Peptide bond + H
= mRNA codon
= tRNA anticodon
= amino acid
One tRNA-amino acid complex remains. It mlJst occupy the A site of the ribosome in order to bind with its codon. Consequently, the dipepMe must move to the right.
- Slide the mRNA to the right (so that tRNA2 is on the P site) and fit the third mRNA codon and tRNA anticodon to form a peptide bond, cre,ating a model of a tripeptide. At about the same time that the second peptide bond is forming, the first tRNA is released from both the mRNA and the first amino acid. Eventually, it will picK up another specific amino acid.
a. What amino acid will tRNA1 pick up?
pairing. On Figure 14-8, attach letters to the odel pieces indicating the composition of your double-stranded DNA model. 5'
Figure 14-8: Drawing of double strand of DNA.
a. What do you notice about the direction in which each strand is running? (That is, are both 5' carbons at the same end of the st ands?)
b. Does the second strand of your drawing how this?
In life, the purines and pyrimidines are joine together by hydrogen bonds. Note again that the sugar backbone is linked by phosphate grou s.
- Examine a three-dimensional model of DNA Notice that the two strands of DNA are twisted into a spiral staircase-like pattern. This is w y DNA is known as a double helix. Identify the deoxyribose sugar, nitrogen-containing bas s, hydrogen bonds linking the bases, and the phosphate groups.
- The second type of nucleic acid is RNA, sho for ribonucleic acid. There are three important differences between DNA and RNA:
a. RNA is a single strand of nucleotides.
b. The sugar is ribose.
c. RNA lacks the nucleotide that contains hymine. Instead, it has one containing the pyrimidine uracil (U) Figure 14-9.
Adenine
Figure 14-4 Double-ringed urines in DNA.
I " - N H
Guanine
The other two nitrogen-containing bases are pyrimidines, specifically cytosine and thymine (abbreviated C and T, respectively). Pyrimidines are single-ring compounds, as shown in Figure 14-5.
Cytosine
NH 0
H C - c-1/
11 Thymine C NH
H 3 C - " - c -
Figure 14-5 Single-ringed yrimidines in DNA.
Although deoxyribose and the nitrogen-co taining bases are organic compounds (contain carbon), the phosphate group is an inorganic ompound, with the structural formula shown in Figure 14-6. 0
HO P - o -
!-
Figure 14-6 Phosphate gr up found in nucleic acids.
The phosphate end of the deoxyribonucleo ide is referred to as the 5' end, because the phosphate group bonds to the 5' carbon atom.
- There are four kinds of deoxyribonucleotid s, each differing only in the type of base it possesses. Construct the other three kinds of deoxyribonucleotides.
