Lecture 3.

2
3.2 transcription & translation

transcription & translation

Hartwell 8e: chapter 8-9, 18

3.2 transcription & translation Phenotypes caused by mutations can tell us about gene function

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 Beadle and Tatum proposed
the “one gene, one enzyme”
hypothesis in the 1940s
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through analysis of
biosynthetic (Arg synthesis)
pathway mutants in
has all amino acids
Neurospora (fungi)

has no amino acids

Auxotroph – mutant strain that cannot grow in minimal

media; requires supplementation (often of an amino acid) 3
 If an enzyme in a pathway is absent, adding its product will restore the normal
phenotype.
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 Check your understanding
You identify four mutations in yeast that arrest growth unless substance X is added to their medium. Through
complementation testing, you determine that these represent four separate genes. You name the yeast
strains X1, X2, X3, and X4. You also isolate the compounds that accumulate in these four strains, calling
them A, B, C, and D. You grow the yeast in medium supplemented with A, B, C, D, or substance X as a
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positive control. Here are the results, where “+” represents growth and “o” represents no growth.

Medium supplemented with:

Strain A B C D sub. X
wild type + + + + +
1 o + o o +
2 o o o o +
3 + + o + +
4 + + o o +

Hint: It’s easier to see the Provide the ordered biochemical pathway that best fits the data
patterns if you rearrange the presented. Indicate both the order of the intermediates (with letters) and
table! the enzymes encoded by the mutant genes in each strain (with numbers).
Lesley Ashmore
 PLEASE check out the exercise I made for you in Top Hat!
3.2 transcription & translation
 Gene expression: the flow of genetic information from DNA via RNA to protein

Central dogma of molecular biology

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 Triplet codons of nucleotides represent individual amino acids

The genetic code is…

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 The genetic code is ________________.

All amino acids except Met and Trp are coded for by more than one codon.
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Is there only one way to

specify every amino acid?
 The genetic code is ________________..

When several codons specify the same amino acid, the first two bases of
the codons are almost always identical.
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 Mutations can be more specifically classified in three ways, based on how they
affect the…
1. sequence of nucleotides in DNA (Module 2)
• Substitution, transition, transversion, insertion, deletion
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2. sequence of amino acids in protein product

• Missense, silent
 Classifying mutations based on the impact on the sequence of amino acids in protein
3.2 transcription & translation product: ________________ mutation

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 The genetic code is ________________..

A single codon never codes for more than amino acid.

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Is there only one way to

interpret a codon?
• Can ACU ever be read
as anything but Thr?

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 The genetic code is ________________.

With a few minor exceptions, all organisms have the same genetic code.
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If you gave different

species the same coding
sequence of mRNA,
would they produce the
same polypeptide?

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 Four characteristics of the genetic code

vRedundant
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vConservative
vUnambiguous
vUniversal
 Yanofsky (1960s): A gene's nucleotide sequence corresponds to the amino acid
sequence of the protein (a gene and its protein are ________________)

trpA
gene
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TrpA

Key observation = Each point mutation affects only one amino acid
• Conclusion: Each nucleotide is part of only one codon!
• Question: How many nucleotides are there in each codon?
 Triplet codons of nucleotides represent individual amino acids

How does DNA with 4 different

nucleotide subunits encode 20 different
amino acids?
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• 1 nucleotide per amino acid = 4 amino

acids
• 2 nucleotide code = 42 = 16 combinations
• 3 nucleotide code = 43 = 64 combinations
 Mutations in different nucleotides may affect the same amino acid
& intragenic recombination can occur between the two mutations

Conclusion:
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 Crick and Brenner (1955) studied frameshift mutations in bacteriophage
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2nd treatment with proflavin caused

________________ suppression!

In other words, created a 2nd mutation in the SAME gene (rllB) that restored wild-type function (revertant!)
3.2 transcription & translation Codons are defined by a reading frame at the beginning of a gene

• Frameshift mutations (for example proflavin exposure) change the grouping of nucleotides as a
result of an insertion or deletion
• A second proflavin-induced mutation can restore the reading frame
 Crick and Brenner’s observations of frameshift mutations (1955) showed that
a codon _________________________________________
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BECAUSE only a net of 0 or 3 (+ or −) mutations restores the reading frame!

(downstream of ATG)
On an exam I might give you similar wt and mutant sequences and ask you to predict which mutant(s) would
yield a functional protein
 Classifying mutations based on the impact on the sequence of amino acids in protein
product: ______________ mutation
3.2 transcription & translation
 Mutations can be more specifically classified in three ways, based on how they
affect the…
1. sequence of nucleotides in DNA (Module 2)
• Substitution, transition, transversion, insertion, deletion
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2. sequence of amino acids in protein product

• Missense, silent, frameshift
 Nirenberg and Matthaei (1961) used synthetic mRNAs and ______________
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translation to figure out which codons represented which amino acids

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 All but the simplest mRNAs leave
some ambiguity
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On an exam I might give you similar data from an alien lifeform

and ask you to give me possible polypeptide sequences
3.2 transcription & translation Nirenberg and Leder (1965) resolved ambiguities with 3-nt mini-mRNAs
 Correlation of polarities in DNA, mRNA, and polypeptide

Coding strand
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• ______________ strand is complementary to mRNA

• ______________ strand (“RNA-like”) has the same polarity and sequence as mRNA
• 5’-to-3’ in the mRNA corresponds to _______to_______ -terminus in the polypeptide
 Sydney Brenner found ______________
mutations, which result in truncated
polypeptides (stop codon = UAG, UAA,
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UGA)

truncated – shorter than normal

 Classifying mutations based on the impact on the sequence of amino acids in protein
product: Nonsense mutation
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 Mutations can be more specifically classified in three ways, based on how they
affect the…
1. sequence of nucleotides in DNA (Module 2)
• Substitution, transition, transversion, insertion, deletion
3.2 transcription & translation

2. sequence of amino acids in protein product

• Missense, silent, frameshift, & nonsense
 Warm-up to this topic

• Take out a piece of paper and spend 1 minute writing down what you
remember about transcription
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 Initiation: The beginning of transcription in prokaryotes requires ______ factor
binding to a ______________
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Only prokaryotes
3.2 transcription & translation Elongation: Constructing RNA copy of the gene

Like DNA polymerase, RNAP obeys the laws of base pairing (but remember that RNA uses UTP instead of TTP)

• (b): (2): ©Professor Oscar Miller/SPL/Science Source

 The ____________ transcript is the single strand RNA resulting from transcription

Coding strand
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• Template strand of DNA is complementary to mRNA

• Coding or “RNA-like” strand of DNA has the same polarity and sequence as mRNA
• 5’-to-3’ in the mRNA corresponds to N-to-C-terminus in the polypeptide

If I give you a template or coding strand of DNA for transcription, you should be able to give me the aa sequence
3.2 transcription & translation
Termination: The end of transcription in prokaryotes

Terminators are RNA sequences that signal the end of transcription

• Two kinds of terminators in bacteria: extrinsic (require rho factor) and intrinsic (don’t require
additional factors)
• Usually form hairpin loops (intramolecular H-bonding)
 In eukaryotes, to make a mature mRNA, the primary transcript must be
processed with a _______cap…
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Gm = methylated guanine
 In eukaryotes, to make a mature mRNA, the primary transcript must be processed
with a 5’ Gm cap & _______ tail…
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The polyA tail is required

for translation and
affects mRNA stability
 In eukaryotes, to make a mature mRNA, the primary transcript must be processed
3.2 transcription & translation with a 5’ Gm cap & 3’polyA tail and have _______ removed
3.2 transcription & translation Three short sequences in the primary transcript determine where splicing occurs

Splice donor Branch site Splice acceptor

3.2 transcription & translation Splicing mechanism: Two sequential cuts remove an intron
 Splicing is catalyzed by a complex of proteins (_______) + snRNAs
3.2 transcription & translation (_____________) called the spliceosome

(bottom): ©Dr. Thomas Maniatis, Thomas H. Lee Professor of Molecular and Cellular Biology, Harvard University

Ribozymes are RNA molecules that act as enzymes to catalyze specific biochemical reactions
 ______________ splicing produces different mRNAs from the same primary transcript
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 Warm-up to the next topic

• Take out a piece of paper and spend 1 minute writing down what you
remember about translation
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 Translation is process of nucleotide (nt) mRNA sequence directing amino acid
assembly into correct polypeptide
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 Transfer RNAs (_______) mediate translation of mRNA codons to amino acids

• Binds to codon in mRNA

• Carries an amino acid corresponding
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to codon
 Aminoacyl-tRNA synthetases
catalyze attachment of tRNAs
to specific amino acids,
creating a _____________ tRNA
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3.2 transcription & translation The tRNA _____________ base pairs with an mRNA codon (they’re antiparallel!)
 How many tRNAs do you think
there are? One per codon?
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• 43 = 64 codons for 20 aa + start/ stop

• >1 codon per aa = _____________ in genetic code
• <45 different tRNAs
3.2 transcription & translation _____________ enables some tRNAs to recognize more than one codon

Flexibility in base pairing between 3’ nucleotide in codon and 5’ nucleotide in anticodon

3.2 transcription & translation Ribosomes (rRNA = ribozymes!) are sites of polypeptide synthesis
3.2 transcription & translation Different parts of a ribosome have different functions
 Translation occurs in 3 stages

1. Initiation stage – initiator codon is AUG near 5’ end of mRNA

2. Elongation stage - amino acids are added to growing polypeptide
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3. Termination stage - polypeptide synthesis stops at the 3’ end of the reading frame
 Translation of mRNAs on ribosomes: Initiation phase in prokaryotes

50S
Prokaryotes
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stabilized by IF2

Will an AUG also initiate transcription if it’s somewhere in the middle of an mRNA?!
 Translation of mRNAs on ribosomes: Initiation phase in eukaryotes

Eukaryotes
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 Translation of mRNAs on ribosomes: Elongation phase
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assisted
by EF-Tu

assisted
by EF-G
+ GTP toward 3’ end of mRNA

EF stands for Elongation Factor

 Complex of several ribosomes translating from same mRNA is a _____________;
allows simultaneous synthesis of many polypeptide copies from a single mRNA
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 Translation of mRNAs on ribosomes: Termination phase
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_____________ factors (not tRNAs) bind to the stop codon, triggering release of
ribosomal subunits, mRNA, and polypeptide
 Posttranslational processing: Enzymatic cleavage may remove an amino acid,
split a polyprotein, or activate a zymogen
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 Posttranslational processing: Addition of chemical constituents may alter protein
structure, activity, or localization
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