Video link below covers how DNA is replicated https://www.youtube.com/watch?v=689sbFTpQOk
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BIOL& 160 Clark College
Biology 160 Lab Module 9 Name __________________
DNA Replication, Transcription, and Translation
Learning Outcomes
Upon successful completion of this lab, you should be able to demonstrate:
An understanding of the complementary, anti-parallel structure of double-stranded DNA.
The process of DNA replication, including names of all enzymes, structures, and the overall procedure. Be able to distinguish between processes of the leading vs lagging strands, and understand how these processes relate to the anti-parallel structure of DNA.
An understanding of the Central Dogma: DNA codes for RNA which codes for Protein
An understanding of the process and products of transcription.
An understanding of the process and products of translation, including the ability to translate a messenger RNA to produce a polypeptide (protein).
A DNA molecule is made of two strands that “complement” each other in the sense that the molecules that compose the strands fit together and bind to each other, creating a double-stranded molecule that looks much like a long, twisted ladder. Each side rail of the DNA ladder is composed of alternating sugar and phosphate groups. The two sides of the ladder are not identical, but are complementary. These two backbones are bonded to each other across pairs of protruding bases, each bonded pair forming one “rung,” or cross member. The four DNA bases are adenine (A), thymine (T), cytosine (C), and guanine (G). Because of their shape and hydrogen bonding sites, the two bases that compose a pair always bond together: A always binds with T, and C always binds with G.
Your instructor might give you an activity to explore the molecular structure of DNA using puzzles, models, or pen-and-paper exercises. Make sure you use these activities to reinforce your understanding of the complimentary and antiparallel structure of DNA.
The particular sequence of bases along the DNA molecule determines the genetic code. Therefore, if the two complementary strands of DNA were pulled apart, you could infer the order of the bases in one strand from the bases in the other, complementary strand. Because the complimentary strands run in opposite directions from each other, the 5’ end of one strand is the 3’ end of the complimentary strand. For example, if one strand has a region with the sequence 3’-AGTGCCT-5’, then the sequence of the complementary strand would be 5’-TCACGGA-3’. Please note: we “read” DNA 5’ to 3’, and so does the machinery that opens and replicates DNA. Helicase unzips the complimentary strands traveling 5’ to 3’ on one of the strands, while another Helicase is unzipping in the opposite direction on the other strand of DNA (like 2 zippers starting in the middle and unzipping in opposite directions away from each other) to make a replication bubble. Each end of the replication bubble is a replication fork. If a new DNA strand is being constructed from left to right it is being constructed 5’ to 3’, and you know that the original DNA is 3’ to 5’ left to right. One of the 2 newly-created DNA strands can simply be created 5’-3’ continuously with DNA Polymerase going away from Helicase – this is the Leading Strand. However, on the other strand (at the same replication fork), DNA Polymerase has to jump back and forth because it can only move 5’-3’ but moving away from Helicase on its strand is the wrong way: 3’-5’. This is the Lagging Strand. The small sections of new DNA created are Okazaki Fragments, joined by DNA Ligase.
Question 1: fill in the blanks below to create a complimentary and antiparallel sequence of DNA (the Template strand is the existing DNA, the Coding strand is the new DNA):
DNA 3’- G _ T G A _ T _ G A C _ A _ T -5’ Coding
... _ G A _ _ G _ A _ _ G T _ C _ ... Template
In order for an organism to grow, develop, and maintain its health, cells must reproduce themselves by dividing to produce two new daughter cells, each with the full complement of DNA as found in the original cell. DNA replication is the copying of DNA that occurs before cell division can take place (see ‘DNA replication’ Figure 1 below).
Figure 1. DNA Replication. The copying of DNA.
To learn more about this process of DNA replication, watch the following video:
https://www.youtube.com/watch?v=TNKWgcFPHqw
Your instructor may have you model the process of DNA replication using puzzle kits or models.
After you have gained an understanding of DNA replication, complete the following diagram using the following terminology list and referring to the image, above:
Helicase
RNA Primase
DNA Polymerase (3 times)
DNA ligase
Okazaki Fragment (3 times)
Leading Strand
Lagging Strand
DNA Replication Review Questions:
In what direction does DNA polymerase read the template DNA strands?
In what direction are the new DNA strands built?
What enzyme forms Okazaki fragments?
What enzyme joins Okazaki fragments?
Explain the relationship between the replication fork(s) and the replication bubble?
Why does DNA have to be replicated?
Part 2: Protein Synthesis - Transcription and Translation Review the process of protein synthesis (transcription and translation) from your classroom notes, textbook sections, or your instructor may play a video for you. Your instructor might also have you model the process using puzzles, models, or paperwork exercises. Use these activities to reinforce your understanding of the two steps (transcription / translation), as well as how they are affected by the Complimentary and antiparallel nature of DNA, and the rules of DNA – RNA base pairing. SUMMARY Figure 2 (below). The making of a protein following the instructions encoded in DNA, via the processes of Transcription and Translation. DNA holds all of the genetic information necessary to build a cell’s proteins. The nucleotide sequence of a gene is ultimately translated into an amino acid sequence of the gene’s corresponding protein.
Figure 3 (at right). The Genetic Code for translating each nucleotide triplet, or codon, in mRNA into an amino acid or a termination signal in a nascent protein.
Use the following worksheet and the figure 3 (preview page) to explore the processes of protein synthesis:
Note: The template strand is the DNA coding strand which is used to create the (complimentary) mRNA. The non-template strand (the complimentary DNA to the coding strand) is essentially the mRNA message, except it has the nucleotide T (DNA) instead of U (RNA). So, the mRNA looks just like the non-template strand of DNA (substitute U for T).
Transcription: DNA coding for RNA
DNA non-template strand sequence: 5’ __G_ _C_ _A__ ____ ____ ____ ____ ____ ____ _G__ _G_ _T_
DNA template strand sequence: 3’ C G T C C A C G T C C A 5’
mRNA strand sequence: _G_ _C_ _A__ ____ ____ ____ ____ ____ ____ _G_ _G _ _U__ 3’
Separate the DNA template and the mRNA strands. In a cell, the mRNA strand would undergo some “editing” including the removal of introns (RNA not needed in the message), addition of the 5’ methyl (CH3) cap and a 3’ poly-A tail. Then it would leave the nucleus, and the DNA strand would rejoin with its complementary half by reforming the hydrogen bonds between complimentary base pairs.
Translation: RNA coding for Protein (polypeptides)
In the cell, the mRNA leaves the nucleus through a pore in the nuclear membrane. Once in the cytoplasm, it attaches to a ribosome and is “read” three base pairs (1 codon) at a time to determine the appropriate amino acid that should be inserted in the growing protein. A block of three consecutive nucleotides in the mRNA is called a codon. The ribosome (which is made of ribosomal RNA or rRNA) is comprised of two subunits named the 40S (“small”) subunit and the 60S (“large”) subunit (the “S” as a unit of weight measurement). The transfer RNA (tRNA) function to bring specific amino acids to the ribosome to be used in building the specific protein. Each tRNA has a three base RNA sequence on one end called the anticodon and a sequence CCA on the other which serves as an attachment site for the tRNA’s specific activating enzyme. The activating enzyme is responsible for catalyzing the chemical reaction of joining the amino acid to the tRNA. The anticodon on the tRNA is complementary to the codon on the mRNA, which explains how the correct amino acid is brought to the site of translation. NOTE: Always use the mRNA codons to find the correct amino acid on the Genetic Code (or, Amino Acid look-up table), not the tRNA anti-codons.
Template DNA base sequence: 3’ C G T C C A C G T C C A 5’
mRNA codons: (5’ to 3’) ______ ______ ______ ______
tRNA anticodons: ______ ______ ______ ______
Amino acid sequence: ______ ______ ______ ______
6. In the previous lab on DNA replication, we discussed that errors can occur during the replication process. Typically, these errors are corrected by proofreading enzymes, but sometimes mistakes persist. A mistake that results in a single base change is called a point mutation and may affect the amino acid composition of the resulting protein.
a) How would the protein have been different if the DNA template sequence was A-G-T-C-G-A-C-C-T-C-C-G? Note: Use the table of the genetic code included earlier in this lab:
Template DNA base sequence: 3’ A G T C G A C C T C C G 5’
mRNA codons: (5’ to 3’) ______ ______ ______ ______
tRNA anticodons: ______ ______ ______ ______
Amino acid sequence: ______ ______ ______ ______
b) Do all point mutations cause a different amino acid to be inserted?
c) Notice from the table of the genetic code (included at the end of this lab) that 61 codons represent the 20 different amino acids. Why do you think it is advantageous, from a genetic perspective, to have this redundancy (i.e. the same amino acid is represented by more than one codon)?
7. Once translation is complete, the protein, the mRNA, and the two subunits of the ribosome separate. In a cell, the resulting protein would be passed into the endoplasmic reticulum where it would undergo further modifications before being used within the cell or exported to other locations within an organism.
8. If the base sequence on a template strand of DNA was TACTATGCCATT, what would be the sequence of the codons on the mRNA transcribed from this DNA?
5’ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ 3’
9. What would the sequence of bases of the anti-codons on the tRNA be?
_____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____
10. What would be the sequence of amino acids that would be translated from this DNA?
_________ _________ _________ _________
11. If a mistake (point mutation) was made during replication of this strand so that the new DNA strand had the base sequence TACACAGCCATT, what would be the resulting change in the protein built from this strand?
_________ _________ _________ _________
12. Complete the following table using the information provided:
| Non-Template DNA Strand | TAC | ||||
| Template | GGG | ||||
| mRNA (5’ to 3’) | CCU | ||||
| tRNA anti-codon | UCG | ||||
| Amino Acid | Leucine |
13. What is the difference, with respect to transcription, between the template and non-template DNA strands?
14. What are the DNA-DNA rules of complementary base pairing?
15. What are the DNA-RNA rules of complementary base pairing?
16. What are the RNA-RNA rules of complementary base pairing?
17. Distinguish between a codon and an anticodon. How are they similar? How are they different?
18. What are the functions of the mRNA, tRNA, and rRNA in translation.
19. In the genetic code table, three codons (UAA, UAG, and UGA) are associated with “STOP”. There are no tRNAs with anti-codons corresponding to these mRNA “STOP” codons. What does this mean with regards to translation?
20. If an amino acid is specified but not the specific mRNA codon, does it matter which codon you choose to code for that amino acid?
DNA Replication and Transcription / Translation Review Questions:
Fill in the blanks – either the DNA sequence, the mRNA sequence, or the amino acid sequence.
DNA 5’- G C A A T G G G T A C A C A A T G A C G -3’ Coding
3’- C G T T A C C C A T G T G T T A C T G C -5’ Template
mRNA ... _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ -3’
DNA 5’- G C A A T G G G T A C A C A A T G A C G -3’ Coding
3’- C G T T A C C C A T G T G T T A C T G C -5’ Template
mRNA ... _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ -3’
Protein Met ___ ___ ___
DNA 5’- G G A _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ -3’ Coding
3’- _ _ T _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ -5’ Template
mRNA 5’- G G A _ _ C G G G U G C A U U A A C C G ...
Protein Met ___ ___ ___
What are the components of a DNA nucleotide?
What are the four nitrogenous bases in DNA molecules?
What are the components of an RNA nucleotide?
What are the four nitrogenous bases in RNA molecules?
What are the percentages of thymine, cytosine and guanine in a DNA molecule that is 30% adenine?
What is meant by the “central dogma” of biology?
What part of the Central Dogma represents transcription? Which represents translation?
What are the effects of mRNA base substitutions on a protein?
What are the effects of an addition or deletion of an mRNA base on a protein?
Where in a eukaryotic cell is DNA found?
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