Question

Translation

Overview:

The purpose of this activity is to help the students to understand how replication, transcription, and translation are connected. Students will use a sequence from a bacterial gene that confers resistance to antibiotics (carbapenems). They will be asked to apply the knowledge obtained in the class lecture to (1) find the promoter in the sequence, (2) determine the amino acid sequence of a fragment of the polypeptide, (3) "reverse translate" a fragment of the polypeptide, and (4) identify mutations in a fragment of the polypeptide.

Intended students population:

This activity is based on basic concepts in Introduction to Biology for science and non-science students.

Learning outcomes:

After this exercise students will be able tor

1. Explain why the flow of genetic information is DNA \(\rightarrow\) RNA \(\rightarrow\) polypeptide.

2. Identify the promoter sequence in a bacterial gene.

3. Apply the genetic code to determine the \(\mathrm{mRNA}\) and polypeptide sequence from a given DNA sequence.

4. Identify different types of mutations in an mRNA sequence and explain their consequences in the translation process.

\begin{tabular}{l}

Vocabulary \\

DNA replication & Gene & \\

Transcription & Genetic code & Mutations \\

Translation & Codon & Promoter \\

mRNA transcript & Polypeptide & Reading frame \\

Wobble pairing & & Polypcptide \\

\hline

\end{tabular}

Prior Knowledge:

Before conducting this activity the instructor should introduce the concepts presented here during the lecture. For the benefit of the students, before this activity you could have them watch a short vidico that explains the \(\mathrm{~ p r o c e s s ~ o f ~ p r o t e i n ~ s y n t h e s i s ~ u s i n g ~ a ~ b a k e r y ~ a n a l o g y ~ ( h t t p s : / w w w . y o u t u b e ~ c o m / w a t c h ' v - m N x Q P m Q L ~}\) As an alternative, you coukd use the "small town library analogy" available at the Genomic Analogy Model for \(\mathrm{~ E d u c a t o r s ~ ( G . A . M . E ) ~ w e b s i t e ~ f r o m ~ P u r d u e ~ U n i v e r s i t y ~ ( h t t p d / w w w ~ y d a e ~ p u r d u e ~ e d u / g a m e l l e s s e n}\)

Students need to know the process of transcription and translation, what is a mutation, and how to read the genetic code table.

Materials needed:

Genetic code table

Escherichia coli promoter sequence

Class textbook

Promoter region

1) Using the bacterial DNA sequence that the instructor gave you:

a. Identify and underline the promoter region and the start codon.

b. Identify the coding and template strand

c. Transeribe the coding sequence

d. Translate the mRNA sequence

2) Explain the purpose/function of the promoter sequence?

3) What could happen if a gene does not have a promoter sequence?

4) How did you determine the start codon?

5) Why does the cell need to make an RNA molecule?

6) Define "codon" and explain the relationship between the linear sequence of codons on mRNA and the linear sequence of amino acids in a polypeptide.

7) Translate the following m R N A sequences and indicate the type and consequences of mutations.

8) Which of the following mutational changes would you predict to be the most deleterious to gene function? Explain your answers.

a. Insertion of a single nucleotide near the end of the coding sequence.

b. Removal of a single nucleotide near the beginning of the coding sequence.

c. Deletion of three consecutive nucleotides in the middle of the coding sequence.

d. Deletion of four consecutive nucleotides in the middle of the coding sequence.

e. Substitution of one nucleotide for another in the middle of the coding sequence.

9) For the RNA sequence below, indicate the amino acids that are encoded in the three reading frames. If you were told that this segment of RNA was in the middle of an mRNA that encoded a large protein, would you know which reading frame was used? How so?

AGUCUAGGCACUGA

10) One strand of a section of DNA isolated from E. coli reads 5^{\prime} '-GTAGCCTACCCATAGG-3'

a. Suppose that an mRNA were transcribed using the complement of this DNA strand as the template. What would the sequence of the mRNA in this region be?

b. How many different peptides could potentially be made from this sequence of RNA? Would the same peptides be made if the other strand of the DNA served as the template for transcription?

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11) Decide whether of this statement is true or false, and then explain why.

a. Wobble pairing occurs between the first position in the codon and the third position in the anticodon.

12) Use the following fragment of a polypeptide sequence to perform 'reverse translation" to determine the mBNA_{\text {segmence }}

Answer 1

1. - Bacterial DNA sequence : (A) Identify and underline promoter region and start codon

In genetics, a promoter is a region of DNA that initiates transcription of a particular gene. Promoters are located near the transcription start sites of genes, on the same strand and upstream on the DNA(towards the 3' region of the anti-sense strand).

2.

Promoter sequences are DNA sequences that define where transcription of a gene by RNA polymerase begins. Promoter sequences are typically located directly upstream or at the 5' end of the transcription initiation site. RNA polymerase and the necessary transcription factors bind to the promoter sequence and initiate transcription. Promoter sequences define the direction of transcription and indicate which DNA strand will be transcribed; this strand is known as the sense strand.Many eukaryotic genes have a conserved promoter sequence called the TATA box, located 25 to 35 base pairs upstream of the transcription start site. Transcription factors bind to the TATA box and initiate the formation of the RNA polymerase transcription complex, which promotes transcription.

3.A promoter sequence is found upstream of all genes and is the site where RNA polymerase binds for transcription to begin. Without it, transcription via RNA polymerase would fail to produce a viable mRNA with a known start and stop site for the gene to be transcribed into polypeptide.

4.

The promoter always exists upstream the start of the gene. So likely you knew the promoter sequence, then several nucleotides laters, you noticed the sequence TAC. TAC is the RNA complement to AUG, which codes for the amino acid methionine. This codon is also called the "start" codon because it denotes the site where translation is initiated for nearly all eukaryotes.

5.

6.The mRNA base triplets are called codon,and they are customarily written 5' ->'3 direction.These codons are complementary to the template strand and thus identical in sequence to the mRNA except that they have T instead of U.During Translation ,the sequence of codons along an mRNA molecule is decoded ,or translated ,into a sequence are read by the translation machinery in the 5' ->'3 direction along the mRNA.

8.Answer is B

Removal of a single nucleotide near the beginning of the coding sequence

This would  be the most deleterious to gene function because the deletion of single nucleotide sets off all subsequent amino acids that are to be coded for.It is more detrimental if this occurs near the beginning of the sequence,because more incorrect amino acids will be coded for in this scenario than if the deletion had occurred toward the end of the sequence.

9.Reading Frame 1- AGU CUA GGC ACU GA

Ser - Leu - Gly -Thr

Reading Frame 2 - A GUCUAG GCA CUG A

Val - Stop - Ala - Leu

Reading Frame 3- AG UCU AGG CAC UGA

Ser - Arg - His - Stop

If this Segment of RNA was in the middle of an mRNA that encoded a large protein ,the first reading frame would have been used because it is only the one that doesnot contain a stop codon

10.(a) CAUCGGAUGGGUAUCC

(b)if one was to begin  at the start codon (AUG) within the mRNA sequence ,a 2 amino acid polypeptide could be created.If it is assumed that there is a start codon before the given sequence,starting at the first reading frame ,a 5 amino acids polypeptide could be created .From the second and third reading frames,respectively.a 5 amino acids polypeptide and 4 amino acid long polypeptide could be created .Thus 4 different peptides could be made from this single sequence of mRNA.

The same peptides would not be made if the other strand of the DNA served as the transcription template .The Amino acids coded for would be completely different if DNA was used in place of mRNA; therefore ,a completely different peptide would be produced.

11.The statement is false.

Wobble paring occurs between the third position in the codon and third position in the anticodons.The first and second position in the anticodons are on the same surface,but the third position of anticodon does not sit on the same plane ,causing a wobble effect .The codons of the RNA all lay flat and casing the tRNA anticodons to wobble back and forth to match all of them together.

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