GENE EXPRESSION

GSND 5113Q

LEARNING OBJECTIVES:
Understand an overview of gene expression,
how the genetic code operates, and
how mutations affect reading the genetic code.
Emanuel Goldman, Ph.D.
New Jersey Medical School
Rutgers Biomedical Health Sciences (RBHS)
Rutgers, The State University of New Jersey
Department of Microbiology, Biochemistry & Molecular Genetics
International Center for Public Health (ICPH)
225 Warren Street, room E450T
Newark, NJ 07103 USA
Telephone: 973-972-4367
Fax: 973-972-3644 or -8982
Email: [email protected]
"Anything that is true of Escherichia coli must be true of elephants, only more so."
- Jacques Monod, 1954 (awarded Nobel Prize in 1965)
<http://muse.jhu.edu/journals/perspectives_in_biology_and_medicine/v047/47.1friedmann.html>

1. Overview of mechanism of gene expression

a. Flow of information from DNA  RNA  protein.

b. mRNA + ribosomes + tRNA

c. Genetic code

d. Genetic language as an information system

e. There are methods to regulate expression at virtually every

step in the information flow pathway.
Mechanism of Gene Expression

DNA

via RNA polymerase(s)

processing via
& processing
modifying &
enzymes modifying
in enzymes
eukaryotes
rRNA tRNA
+ via
r-proteins aa-tRNA
synthetase
enzymes

mRNA + ribosomes + aa-tRNA

all proteins
(including: RNA polymerase
r-proteins
processing & modifying enzymes
aa-tRNA synthetase enzymes)
 The Genetic Code
2 U C A G 3
UUU UCU UAU UGU U
1 UUC
Phe
UCC UAC
Tyr
UGC
Cys
C
U UUA UCA
Ser
UAA Term UGA Term A
Leu
UUG UCG UAG UGG Trp G

CUU CCU CAU His CGU U

CUC CCC CAC CGC C
C CUA
Leu CCA
Pro
CAA Gln CGA
Arg
A
CUG CCG CAG CGG G

AUU ACU AAU Asn AGU Ser U

AUC Ile ACC AAC AGC C
A AUA ACA
Thr
AAA Lys AGA Arg A
AUG Met ACG AAG AGG G

GUU GCU GAU GGU U

Asp
GUC GCC Ala GAC GGC C
Gly
G GUA
Val GCA GAA Glu GGA A
GUG GCG GAG GGG G

U, uracil; C, cytosine; A, adenine; G, guanine. The first code letter is denoted from the left column
(1), the second code letter from the top row (2) and the third code letter from the right column (3).
Triplet codons coding for the same amino acid are bracketed. Standard 3 letter abbreviations are
used for amino acids. “Term” is used as the abbreviation for termination (i.e., stop) codons.
How was the genetic code deciphered?
• Accomplished by in vitro protein synthesis (at high Mg++) with synthetic
polyribonucleotides (Nirenberg & Matthaei; Khorana),
and by "triplet binding" (Nirenberg & Leder) experiments.
• Later confirmed by DNA and/or RNA sequencing of genes whose protein
products had been independently sequenced.
• Nearly universal (some variations in mitochondria).
• Stop codon UGA occasionally read as selenocysteine.
• "Recoding" events: signals in mRNA that cause the protein synthesis
machinery to behave anomalously.

• Poly(U) directs synthesis of polyphenylalanine in a

cell-free translation system (Nirenberg & Matthaei, 1961)
• Poly(A) promotes synthesis of polylysine,
• Poly(C) promotes polyproline synthesis

PolyUC codes for a Ser-Leu repeating polypeptide

Polynucleotide 5’------UCUCUCUCUCUCUCUCUC------ 3’
In vitro translation

Polypeptide Ser–Leu–Ser–Leu–Ser-Leu
Mild acid hydrolysis

Ser-Leu
 In Vitro Protein Synthesis
• E. coli cell pellets broken open mechanically in the cold
(originally ground with alumina in a mortar and pestle;
later via French Pressure cell at ~5,000 pounds/square inch)
• Lysate centrifuged cold (sometimes with DNase) at 10K to remove debris
• Lysate centrifuged cold at 30,000 x g; supernatant (dialyzed) = "S30"
quick-frozen in aliquots & stored at –70 oC
(this extract is capable of supporting protein synthesis in vitro)
• Further fractionation by ultracentrifugation at 100,000 x g yields an "S100" and
a crude ribosome pellet. Pellet can be salt-washed to remove and purify factors.
These components (dialyzed) can be mixed to support protein synthesis in vitro.
• In vitro protein synthesis reactions contain the following ingredients:
- S30 or S100 + portion of ribosome pellet (+ factors if washed)
- Buffer containing Mg++ & K+ at neutral pH and reducing conditions (e.g., mercaptoethanol)
- A mixture of all 20 amino acids
( if assayed by radioactive incorporation, 19 cold and one radioactive or other variations)
- ATP + GTP + energy regenerating source (e.g., phosphoenolpyruvate)
- UTP and CTP if making mRNA
- Source of mRNA
(including coupled transcription-translation from DNA template + RNA polymerase)
- Reaction can be supplemented with additional tRNA or other reagents but this is not required
(S30s and S100s contain tRNAs and aminoacyl-tRNA synthetases)
Other widely used in vitro protein synthesis systems derived from reticulocytes and also from wheat germ
 Variations in mitochondrial genetic code
Phylum or UGA AUA AAA AGR CUN UAA
Subphylum (Stop) (Ile) (Lys) (Arg) (Leu) (Stop) Examples

Vertebrates Trp Met -- Stop -- -- Human, bovine, rat,

mouse, chicken, frog
Tunicates Trp Met -- Gly -- -- Sea Pineapple (Halocynthia roretzi),
Sea squirt (Pyura stolinifera)
Echinoderms Trp -- Asn Ser -- -- Sea urchin, starfish

Arthropods Trp Met -- Ser -- -- Drosophila, mosquito,

honeybee
Molluscs Trp Met -- Ser -- -- Squid, Mussel (Mytilus edulis)

Nematodes Trp Met -- Ser -- Roundworm of pigs (Ascaris suum),

--
Caenorhabditis elegans
Platyhelminths Trp -- Asn Ser -- Tyr? Liver fluke (Fasciola hepatica),
Planaria (non-parasitic flatworms)
Coelenterates Trp -- ND -- ND ND Sea anemone (Metridium senile)
Hydra
Yeasts Trp Met -- -- Thr -- Saccharomyces cerevisiae,
Torulopsis glabrata
Euascomycetes Trp -- -- -- -- -- Aspergillus nidulans,
Neurospora crassa
Protozoa Trp -- -- -- -- -- Trypanosoma brucei,
Paramecium
There are also a few other minor variations from the universal genetic code in some bacterial and one-cell
eukaryotes. For example, in several yeast Candida species, CUG codes for serine instead of leucine. In many
mycoplasma species, UGA codes for tryptophan instead of stop.
See also: Watanabe K, Yokobori S. (2011) tRNA Modification and Genetic Code Variations in Animal Mitochondria. J Nucleic Acids 2011:623095
GENETIC LANGUAGE AS AN INFORMATION SYSTEM
English Language Genetic Language
letters in alphabet 26 (a to z) 4 (A,T,G,C)
(representing specific chemicals in DNA)
word length varies 3 letters only (triplets)
words in dictionary >100,000 61 (out of 64 possible triplets)
meaning of words complex, varied 20 specific meanings, 41 synonyms
(each of the 20 amino acids which make up proteins)
punctuation ,:;-("'[?!. . (TAG, TAA, TGA)
(major usage) (remaining 3 of the 64 possible triplets)
spaces used between words no spaces between words
direction of reading left to right by convention, presented left to right
(in chemical terms, 5' to 3')
text begins with capital letter with 1 of the 61 words
(generally, ATG)
each text contains many sentences one sentence (= one gene)
sentences contain from a few to perhaps from as few as 30 or so words up to
up to 40-50 words 1000 or more

sample sentence in English language:

"To be or not to be: that is the question."

sample (very short) sentence in Genetic language:

ATGCTGAAACGTCCGTGGATCAAGTTCGGCTAA
translation of this sentence into a fragment of protein (i.e., a 10 amino acid long peptide):
methionine-leucine-lysine-arginine-proline-tryptophan-isoleucine-lysine-phenylalanine-glycine-.
(note two synonyms for lysine in this example, AAA and AAG)
 Coding and Template Strands of DNA
Elongation RNA
Coding strand of DNA
nucleotides
RNA polymerase

3’ 3’ end

5’

5’ Direction of transcription
Template strand
of DNA
Newly made RNA

All RNA polymerases are dependent upon a DNA template in order to synthesize
RNA. The resultant RNA is, therefore, complementary to the template strand of
the DNA duplex and identical to the non-template strand. The non-template
strand is called the coding strand because its sequences are identical to those of
the RNA product (with the exception that ‘T’ in DNA is ‘U’ in RNA).
Modified from a slide at https://exploreable.wordpress.com/2011/05/16/from-dna-to-rna-the-process-of-transcription/
Genetic engineering is the chemical equivalent of using the
"cut and paste" function of your Word Processor (or scissors and
scotch tape on actual paper) to cut a gene from one location and
paste it in another.

For example, the gene coding for "insulin" in a human cell can be cut
out by chemical scissors (enzymes) and pasted by chemical tape
(other enzymes) into the DNA of bacteria. Under the right
conditions, the bacteria will then be able to make human insulin.

Because the gene is a unit of information, the information itself can

be used chemically, and the original physical source of the
information is no longer needed.
 HOW MUTATIONS AFFECT READING THE GENETIC CODE

Review and Special Terminology

• Definitions:
• Genotype (genetic potential)
• Phenotype (observable characteristics)
• Missense, nonsense and frameshift mutations (see Figures below)
• Temperature-sensitive mutations (usually missense; proteins)
• Suppression of mutation (several mechanisms, covered later)
• Auxotrophs (nutritional requirement due to mutation) and prototrophs

• Mutagens: DNA damage, resulting in changes in sequence

 EFFECT OF BASE SUBSTITUTIONS ON PROTEINS

MetThrMetIleThrAsnSerArgGlySer Wild type

AUGACCAUGAUUACGAAUUCCCGGGGAUCC
(1) MetThrMetIleThrAsnSerArgGlySer U C ; Ile Ile
AUGACCAUGAUCACGAAUUCCCGGGGAUCC no change in aa
*
(2) MetThrMetIleThrAsnAlaArgGlySer U G ; Ser Ala
AUGACCAUGAUUACGAAUGCCCGGGGAUCC aa change
*
(3) MetThrMetIleThrAsnSerArgStp G U ; Gly Stop (Stp)
AUGACCAUGAUUACGAAUUCCCGGUGAUCC termination of protein
*

(1) Results in a “silent mutation”: no change in phenotype. Heterogeneity within a population is a consequence of
changes like these, and can be detected as “Restriction Fragment Length Polymorphisms” (RFLPs).
(2) Can result in a missense mutation, if the amino acid change affects function of the protein. Can also result in a
silent mutation if the amino acid change does not affect function of the protein.
(3) Results in a “nonsense” mutation, causing premature termination of protein synthesis. Almost always causes
loss of function of the protein (exception if the mutation is very close to the normal end of the coding region).
 Example of base substitution mutation
Sickle cell disease is caused by a mutation in the gene for the hemoglobin beta chain at the
position specifying amino acid number 6 of the protein (the starting methionine residue, which is
removed from the mature protein, is not counted in the numbering).

The DNA coding strand, mRNA, and protein sequences over this region for both wild type and
sickle cell are shown below:

cagacacc atg gtg cat ctg act cct gag gag aag... wild type DNA
CAGACACC AUG GUG CAU CUG ACU CCU GAG GAG AAG... wild type mRNA
Met Val His Leu Thr Pro Glu Glu Lys... wild type globin

cagacacc atg gtg cat ctg act cct gtg gag aag... sickle cell DNA
CAGACACC AUG GUG CAU CUG ACU CCU GUG GAG AAG... sickle cell mRNA
Met Val His Leu Thr Pro Val Glu Lys... sickle cell globin

The A to T change in codon 7 (boxed) converts the wild type glutamic acid residue to a mutant
valine residue. Although the mutation is harmful and life threatening in a homozygote, the Glu6Val
substitution has been retained in the human population in malaria endemic regions because
heterozygotes are relatively resistant to malaria.
 EFFECT OF FRAMESHIFT MUTATIONS ON PROTEINS
The following sequence: MetThrMetIleThrAsnSerArgGlySer can be read in 3 frames:
AUGACCAUGAUUACGAAUUCCCGGGGAUCC
Frame 1 Met Thr Met Ile Thr Asn Ser Arg Gly Ser
(correct) AUG ACC AUG AUU ACG AAU UCC CGG GGA UCC
Frame 2 Stp Pro Stp Leu Arg Ile Pro Gly Asp Pro
(incorrect) A UGA CCA UGA UUA CGA AUU CCC GGG GAU CC
Frame 3 Asp His Asp Tyr Asp Phe Pro Gly Ile
(incorrect) AU GAC CAU GAU UAC GAA UUC CCG GGG AUC C
Three frames are possible: note the drastic effects of an incorrect frame on amino acid sequences. Normally
ribosomes initiate protein synthesis at the first AUG codon and read succeeding codons contiguously.
Therefore only Frame 1 is used. The other 2 frames are shown to illustrate how 3 frames are possible.
Frameshift mutations:
30 nt Met Thr Met Ile Thr Asn Ser Arg Gly Ser Wild type
AUG ACC AUG AUU ACG AAU UCC CGG GGA UCC
31 nt Met Thr Met Ile Asp Asp Phe Pro Gly Ile +1 frameshift; protein sequence
AUG ACC AUG AUU GAC GAA UUC CCG GGG AUC C change (underlined)
* (G inserted)
29 nt Met Thr Met Ile Arg Ile Pro Gly Asp Pro -1 frameshift; protein sequence
AUG ACC AUG AUU AGA AUU CCC GGG GAU CC change (underlined)
* (C lost)
30 nt Met Thr Met Ile Arg Ile Leu Arg Gly Ser -1, +1 double frameshift can restore
AUG ACC AUG AUU AGA AUU CUC CGG GGA UCC original frame although aa between the
* (-1) * (+1) frameshifts are changed (underlined)
(C lost) (U inserted)

Insertion or deletion of a single nucleotide (+1 or -1) causes a frameshift. Note that ribosomes initiate synthesis
at AUG but because of the gain or loss of a single nt the frame is automatically shifted and wrong aa are assembled.
As seen above, frameshifts can garble messages completely and destroy a gene. Sometimes a second frameshift of
opposite sign (+ versus -) can restore a frame, and under some conditions, restore function as well.
Effect of Macromutations
Large deletions
Large insertions
Rearrangements

All can lead to loss, gain, frameshifting and scrambling of blocks of codons

Usually destroy gene function, but not always

 Identification of the ‘Amber’ Nonsense codon
Trp
UGG
Lys Ser
AAG, AAA UCG, UCC, UCA
UCU, AGU, AGC

Gln Amber Tyr

CAG, CAA ??? UAU, UAC

Glu Leu
GAG, GAA UUG, UUA, CUU
U U U CUC, CUA, CUG
C C C
A A A
G G G
Identification of the amber nonsense codon as UAG. A Pho- mutant of E. coli (lacking the enzyme alkaline phosphatase) was isolated; this
mutant carried an am mutation in its phoA gene. From this Pho- mutant, a set of Pho+ reverse mutants was isolated whose recovery of
alkaline phosphatase was not attributable to suppressor mutations. Amino acid sequence analysis of the alkaline phosphatase protein of
these reverse mutants showed that they carried the variety of amino acid replacements shown in this diagram at the same polypeptide site
at which the wild-type alkaline phosphatase carries a tryptophan residue. (Out of 449 residues, 3 are Trp, at positions 109, 220, and 268.)
Each amino acid is represented here by its synonymous codons, and those codons that are connected to UAG by a single base change are
underlined. It is evident that UAG is the only nucleotide base triplet that has this connection to at least one of the codons for each of the
seven amino acid replacements found. [after A. Garen, Science 160, 149 (1968).]
Historical note: Amber mutations were the first set of nonsense mutations to be discovered, isolated by graduate student Harris Bernstein ...
(whose last name means "amber" in German), [who] had been offered the reward of having any discovered mutants named after himself
<http://en.wikipedia.org/wiki/Stop_codon>.