Lab 9 - Transcription and Translation (30 points)
Name:_______John Gibson____________________________
Section # (801-817)______807__________________
Instructor Name:________George Gikas___________________
(3 pts) What was the purpose of the dish detergent, salt and ethanol in the DNA extraction experiment? Describe the purpose of each individually.
Detergent - breaks plasma membrane of strawberry cells. Detergents, such as SDS, are amphipathic with hydrophilic head and single hydrophobic tail. The hydrophobic tail inserts into the membrane phospholipid with double tails and changes conformation from bilayer to micelle, breaking the cell apart and releasing DNA.
Salt - weakens hydrogen bonds between negative DNA phosphate group and protein positive peptide sequences so that DNA can be free to leave the tangle of protein extract.
Ethanol - precipitates out soluble, highly polar, charged DNA by the hydrophobic hydrocarbon part of ethanol. Ethanol is relatively low polar compared to water, which dissolves the freed DNAs that are floating, not condensed. Precipitation condenses DNA into 1 layer in the column.
(3 pts) How is RNA different than DNA? Describe three key differences at the molecular level.
RNA is usually single stranded. DNA is usually double stranded.
RNA has 2’ OH. DNA has 2’ H.
RNA is more reactive with the 2’ OH’s electron lone pair. DNA is less reactive with C-H bond without electron lone pair. Straight C-C and C-H bonds often need bromine compounds with UV light activations to attach a bromine to it to become reactive, unless combustion temperature energy is use.
RNA is unstable, degrading in minutes to hours. DNA is stable over years, decades, centuries, millennia.
(4 pts) What is the protein sequence generated by the mRNA in lab? Be sure to label the N and C termini.
N-Met-Trp-Pro-Arg-His-C
(3 pts) What occurs in the E, P and A sites of the ribosome (be specific for each site)?
A site - tRNA binds to DNA with anticodon, bringing in codon specific amino acid
P site transfers the amino acid on tRNA to the growing peptide by dehydration condensation reaction to form an amide bond between the growing peptide’s C tail and the new amino acid’s N amin group.
E site - it releases used-up, discharged tRNA from mRNA
(2 pts) The stop codon, UGA, sometimes serves another function in the cell. Do some research to determine what else can occur when a UGA codon is reached provided the downstream RNA is the appropriate sequence and structure.
The poly-A tail of mRNA can be in a curled conformation, bound to the 5’ cap of mRNA’s translation initiation factors (eIF4), or bound to other 5’ initiation factors. The two schemes are referred to as cap-dependent and cap-independent.
So, when translation ends at UGA, the ribosome 60S unit is located conveniently at the 5’ end of mRNA, ready to go for another round of peptide synthesis.
(3 pts) The table below contains anti-codons. Fill in the corresponding codon (indicate directionality) and amino acid.
Transcription, Translation Additional Practice
(3 pts) Using the DNA sequence below:
5’-TTAATGACGGCTTACGATAAGTGAGCA-3’
3’-AATTACTGCCGAATGCTATTCACTCGT-5’
Using the lower strand as the template strand, you are going to transcribe the DNA message into messenger RNA.
What direction does RNA polymerase read the DNA?
3’ to 5’
What direction is mRNA synthesized?
5’ to 3’
What is the sequence of the mRNA? Indicate 3’ and 5’ ends.
5’-UUAAUGACGGCUUACGAUAAGUGAGCA-3’
(2 pts) Using your answer from question above, translate the mRNA into protein.
What codon does translation start at?
AUG
Indicate N and C termini on the protein sequence below
N-Met-Thr-Ala-Tyr-Asp-Lys-C
(2 pts) Using the DNA sequence below:
5’-TTAATGACGGCTTGCGATAAGTGACATGCA-3’
3’-AATTACTGCCGAACGCTATTCACTGTACGT-5’
Using the upper strand as the template strand, you are going to transcribe the DNA message into messenger RNA.
What is the sequence of the mRNA? Indicate 3’ and 5’ ends.
3’-AAUUACUGCCGAACGCUAUUCACUGUACGU-5’
, or, equivalently,
5’-UGCAUGUCACUUAUCGCAAGCCGUCAUUAA-3’
(2 pts) Using your answer from question above, translate the mRNA into protein.
C-His-Arg-Ser-Ala-Ile-Leu-Ser-Met-N
, or, equivalently,
N-Met-Ser-Leu-Ile-Ala-Ser-Arg-His-C
(3 pts) Using the mRNA sequence below:
5’-GUCAUGAAUCAUAGGCUUAAGCCGUAGCAC-3’
Work backwards and write out the double stranded DNA sequence that was used to synthesize the above mRNA sequence.
Indicate 3’ and 5’ ends on BOTH strands
Indicate which strand served as the TEMPLATE strand and which strand served as the CODING strand.
5’-GTCATGAATCATAGGCTTAAGCCGTAGCAC-3’ (coding strand)
3’-CAGTACTTAGTATCCGAATTCGGCATCGTG-5’ (templated)
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