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LS1a Fall 2014
Section Week #7
I.
Gene regulation

In positive control, a gene’s expression depends on a DNA-binding protein (an “activator”) to bind
the DNA and turn ON transcription by recruiting RNA polymerase to the promoter.
o

Positive control requires weak promoters whose -10 and -35 elements poorly match the
consensus sequences.
In negative control, a gene’s expression depends on a DNA-binding protein (a “repressor”) to bind
the DNA and turn OFF transcription by preventing RNA polymerase from binding.
Section Activity #1: The lac operon represents a model system of prokaryotic gene regulation. (An
“operon” is a contiguous series of genes that have a related function when expressed in the cell; in
this case metabolizing lactose. All of the genes in an operon are under the control of a single
promoter.) Two proteins, CAP and LacI, regulate the lac operon by influencing whether or not RNA
polymerase can bind to the lac operon’s promoter. A map of the lac operator is shown below:
a. CAP binds to a DNA sequence upstream of the promoter only when levels of a small molecule
called cAMP are high. Transcription from the lac operon promoter is high only when levels of
cAMP are high. Is CAP a repressor or an activator? Is this an example of positive or negative
control? Briefly explain your answer.
b. LacI binds to a region of DNA overlapping with the promoter only when the levels of a small
molecule called lactose are low. When lactose is added to a cell, expression of lac operon
genes is enhanced. Is LacI a repressor or an activator? Is this an example of positive or
negative control? Briefly explain your answer.
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II. Translation
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Three classes of RNA are required for translation:
o mRNA is the informational template.
o tRNA (where “t” = “transfer”) acts as a molecular adaptor that matches amino acids (aa) to
the mRNA code.
o rRNA (where “r” = “ribosomal”) associates with ribosomal proteins to form the ribosome.
A nucleotide triplet (e.g., AGA) in mRNA is called a codon. Each codon encodes one amino acid
(except for stop codons, which do not encode amino acids).
Codons are read consecutively on mRNA from 5’ to 3’.
The mRNA code can be translated in one of three reading frames. The reading frame is typically
established by the first start codon (AUG). Only one reading frame will result in translation of the
correct protein.
Section Activity #2: The reaction in which tRNA is charged with threonine is shown below.
a. What enzyme catalyzes this reaction?
b. Another molecule is required to make this process thermodynamically favorable. Indicate this
molecule and its byproducts in the boxes above.
c. Label the 5’ and 3’ ends of the tRNA in the drawing above. Briefly explain how you can tell.
d. What does the anti-codon do? How many nucleotides does it contain?
(Continued on next page…)
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Section Activity #2: (continued from previous page)
e. Which of the following amino acids, Arg, Trp, Phe, Tyr, and/or Ser, would you expect to be
excluded from the synthesis site of the threonyl aminoacyl tRNA synthetase?
f.
How would you expect the threonyl-tRNA synthetase synthesis site to discriminate between
threonine and valine?
g. What is the function of the editing site? How would you expect the editing site to prevent a
serine from being attached to a threonine-specific tRNA (“tRNAThr”)?
h. If alanine were accidentally attached to a threonine-specific tRNA (“tRNAThr”), would you
expect the ribosome to incorrectly integrate it into a protein?
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Section Activity #3: Shown below is the sequence of an mRNA that encodes a protein in zebrafish.
A large portion of the 3’ untranslated region (from 661-1320) has been removed and is indicated
as “…”. The stop codon has been underlined. The sequence is written 5’ to 3’, and there is a codon
table on the next page.
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61
121
181
241
301
361
421
481
541
601
UAGAUUUAAA
UUUGUUACUG
GUUAUGAUUG
AUUUUUUGGU
CUGUCAGAGC
AGCUGCAGCA
UGUGCAUCAA
CAUCAAGCUG
AGACAAAAAU
UUUCAUUUUC
AAUAUCCAUA
AGACAGCCCC
UUCUUUGUGU
UGGAACUUGG
ACAAGCAAGU
CCAUUUUUUA
CCAAUCUGAC
GAGAAUUUUA
CAGCAUAUAA
UCAAUGAAAA
UGACUAAAAU
UCUUUCAGCA
CAAAAAUUGU
UGCGCGCACU
CAAUAGCGUG
UGCUGGUCAA
CAACAAAUAC
AAUCUCAGAC
UACUGAGUUU
GAACUCAAAU
CAGCACAACA
GAAUCUUCUA
AAUGUUUCAG
UAUACUAUAU
GCACAUUCAC
ACUCUGCGGU
CAACCUCGAG
AAAAAUGAAC
AUCAUCCAGU
GGAAAUGGAA
UUAGAUCAUC
GAAAUGUGUA
CAAGUUUGUG
AUGAAAGGCU
CUACAAUGAU
GUAUUGAACA
GUAUUUCGUG
UCAUUUCAGC
GUUUCUGGGG
CAGAUGAAGC
CACAUCUGAA
UGAACAGCUC
AUUAUUUAUG
UAUGUAUUCU
CUGUCCAGCA
UCUCUGGGCA
GCCAGAAAAA
UAACAAUAAC
AUUUCAGAAA
CAAAAGACUG
AGUGUACUAU
AAUUAAAGGU
CAUCAAGUGU
AAUCCACUUC
CAGAAUCAUU
GUGCUUGGUU
…
1321
1381
GUAGUGCAUU AUAUUUACGU UAUUCAAUUU ACAUUGCAUG AGAUAAACUU UAUGGAUCAU
UAAAAAAAAA AAAAAAAAAA AAAAAAAAAA A…
a. Identify the start site of translation in the sequence above.
b. What are the first two amino acids of this protein (N- to C-terminal)?
c.
Briefly describe how the translated protein would be affected if the following mutations were
made to the mRNA:
i.
U51 is changed to C
ii.
U529 is changed to G
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The ribosome is a large complex consisting of about 1/3 protein and 2/3 ribosomal RNA. Ribosomes
are located in the cytoplasm and consist of two subunits, termed large and small. Both subunits
associate at the start codon of an mRNA transcript to begin the synthesis of protein.

3 sites in the ribosome bind to tRNA
o A-site, binds aminoacyl-tRNA (think: site that accepts the incoming tRNA)
o P-site, binds peptidyl-tRNA
o E-site, the site where a “spent” t-RNA exits
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Elongation happens as follows:
o Step 1: The incoming aminoacyl-tRNA binds to the A-site.
o Step 2: The bond between the C-terminus of the amino acid chain and the tRNA in the P-site
is broken as the amino acid chain makes a new bond to the amino-group of the amino acid in
the A-site. The mRNA advances by three nucleotides, placing the tRNAs in the E- and P-sites.
o Step 3: The spent tRNA is ejected from the E-site and the ribosome is reset to bind another
aminoacyl-tRNA at the A-site.

EF-Tu binds tightly to charged tRNAs when bound to GTP. When a charged tRNA enters the
ribosome, it is still bound to EF-Tu. The peptide bond cannot form until EF-Tu hydrolyzes GTP to
GDP and dissociates from the tRNA, allowing it to completely enter the A-site.

EF-G binds near the A-site and hydrolyzes GTP to GDP to facilitate movement of the mRNA
through the ribosome along with the movement of the tRNAs to the E and P sites.
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Section Activity #4: Your TF will hand you cutouts of ribosomes in various stages during the
translation cycle.
a. Arrange the cutouts of the ribosome in the correct order.
b. On image (A), indicate the region on the messenger RNA transcript that corresponds to the
codons that encode the red and yellow amino acids.
c. What is the color of the amino-terminal amino acid residue?
d. Between which stages from part (a) do the following processes occur:
i.
EF-G hydrolyzes GTP.
ii. EF-Tu hydrolyzes GTP.
iii. A peptide bond forms.
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Section Activity #5: Statement Corrections
Determine whether each statement (a-d) is correct as written. Some of these statements may be
correct and some are completely wrong. If you identify an incorrect aspect to one of the
sentences, write a more concise, accurate, and complete sentence than the one shown.
a. Positive control refers to any situation in which a stimulus increases expression of a gene.
b. Only in prokaryotes is the entirety of an mRNA transcript translated; this is because eukaryotes
have introns that are spliced prior to translation.
c.
Translation will be halted anytime the three nucleotides “UGA” are found together.
d. During the formation of the peptide bond during translation, the entire polypeptide bound to
the tRNA in the P site is attached to the N-terminus of the amino acid attached to the tRNA in
the A site.
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