Genes and Proteins
I. Overview of Protein Synthesis
A. Process: DNA => RNA => Polypeptide => Protein
B. Why a 2-step process?
1. Evolutionary limitation
a. RNA probably preceded DNA
b. Some RNAs can self replicate
2. DNA is more stable (less damage)
3. Productivity: EXAMPLE: can get five times more protein from one DNA gene
being transcribed into 10 copies of mRNA which can be used to make 10
proteins each.
II. Historical Development of Gene-Protein Relationship
A. 1902 - Archibald Garrod
Alkaptonuria : homogentisic acid (HA) is metabolized to maleylacetoacetic
acid. HA builds up in tissues, urine, cartilage and turns them black.
Garrod determined that the disorder was inherited as a recessive trait..
Linked a chemical activity to heredity.
B. 1941 - George Beadle and Edward Tatum
Studied heritability of metabolic mutations in the bread mold, Neurospora
crassa. On minimal medium, it could manufactory all 20 AA, purines,
pyrimidines, and vitamins. Induced mutations with X-radiation then
plated the fugal colonies on minimal and complete medium. Anything
that didn't grow on minimal had mutated. Could then plate up those
grown on complete on minimal plus one nutrient to isolate the function of
the mutated enzyme. B6, B1 plus hundreds more mutants isolated.
Then, bred with wild-type to establish nuclear origin to mutant. Beadle
and Tatum suggested that independent mutants could be found for every
enzymatically control reaction. One "mutation" one enzyme
hypothesis
C. 1944 - The final link was placed when Avery, McCary and McLeod
demonstrated that DNA was genetic material. This must be site of
nuclear mutations. As of 1945: "One DNA gene - one enzyme". Next
Step: analysis of Sickle-Cell Anemia
D. 1949 - James Neel and E.A. Beet demonstrated that Sickle-cell was inherited
as a 2-allele Mendelian trait.
AA - normal
AS - largely unaffected carriers - blood does sickle
SS - Sickling - under low oxygen tension (venous blood)
erythrocytes elongate (sickle). RBC's aggregate and block
capillaries, depriving tissues of oxygen. Crisis may be fatal as
kidneys, brain muscles and lungs affected.
E. 1949 - Linus Pauling, Itano Singer and Wells found that hemoglobins isolated
from normal and sickle-cell migrate in electric field (Electrophoresis) at
different rates. Concluded that a chemical difference existed between
to hemoglobin forms (HbA and HbS).
F. 1954, 1957 - Vernon Ingram found that chemical difference occurred in globin
portion (proteinaceous), not the non-protein heme groups. He found
that Human Hemoglobin contains 4 polypeptides in globin protein; 2
alpha chains (141 AA long) and 2 beta chains (146 AA long). The HbS
had a single AA change in the 6th position of the Beta chain. Valine
instead of Glutamic Acid
G. Implications:
1. Heritable (DNA) mutation affecting a non-enzymatic protein. DNA codes for
proteins, not just enzymes.
2. Affect is at level of polypeptide - "One gene - one polypeptide"
The link was made: DNA determines protein formation. The question now
was HOW? How does the sequence of nucleotides in DNA determine the
sequence of AA in a protein?
Two questions:
(1) Information Transfer - conversion of DNA into mRNA - Breaking the
"Genetic Code". Question degenerates to "how does the
ribonucleotide sequence of mRNA (U,A,G,C) code for amino acid?
(2) The mechanism - Transcription/Translation
III. Summary of the Code
A. Characteristics
1. Linear - sequence of nucleotide bases are linear
2. Triplet Code - each sequence of 3 nucleotide bases specifies one amino acid
[Codon]
3. Unambiguous - each triplet specifies only one amino acid
4. Degenerate - A particular amino acid is specified by more than one triplet.
5. Start and Stops - Initiation and Termination Codons
6. Ordered - degenerate codons for a given amino acid are grouped together
most often varying by only the third base
7. No internal punctuation
8. Non-overlapping
9. Universal - used by almost all Pro- and Eu- karyotes
IV. Deciphering the Code
A. George Gamow (1954): Diamond Code, overlapping DNA code
1. no intermediate - direct DNA template
2. overlapping
B. Arguments against overlapping code
1. Sidney Brenner: Presumed triplet code (fewest number that could code for
20 amino acids). Reasoned that overlap code meant restriction on amino acid
sequencing. For any 3-base sequence, only 16 possible tripeptides (4 in first,
X 4 in third) 24 = 16
2. Mutation - with overlapping code, .1 amino acid would change; yet human
hemoglobin revealed single amino acid changes.
3. Crick 1957 - coding would require H-bonding, which is unlikely between
nucleotides and amino acids. Requires an adapter molecule (tRNA) - would
covalently bind to the amino acid, yet be capable of hydrogen bonding to a
nucleotide sequence. An overlapping code would require that these adapters
overlap - seemed structurally unlikely
C. Francois Jacob and Jacques Monod (1961) - postulated the existence of
messenger RNA (mRNA)
D. Marshall Nirenberg and J. Heinrick Matthaei (1961)
1. Required two things:
a. A cell-free protein-synthesizing system: ATP, CTP, UTP, GTP; Mg++, K+
(stabilizers) Create random RNA fragments!
b. The enzyme Polynucleotide Phosphorylase allowed the production of
synthetic mRNAs. These mRNAs served as templates for polypepetide
synthesis in the cell-free system.
2. Put RNA fragments in with ribosomes, tRNA, amino acids, and enzymes =>
create polypeptides
a. Homopolymers: Assuming triplet, UUU => Phenylalanine
Repeated for AAA => lysine
GGG => didn't work right - molecule folded
CCC => proline
b. Heteropolymers: Ratios of 2 bases: 1A:5C can predict ratio of triplets
3A = AAA = (1/6)3 = 0.4 = Lysine
2A:1C = AAC, ACA, CAA = (2.3) X 3 = 6.9 = Asparagine (2) Glutamine (2)
1A:2C = ACC, CAC, CCA = (11.6) X 3 = 34.8 = Threonine (12) Histidine (14)
3C = CCC = 57.9% = Proline
Defining the combo's using other techniques led to a complete description of
the code.
D. The Triplet Binding Technique
1. 1964 Nirenberg and Philip Leder: Triplet Binding Assay (Specific Sequence
Assignments for the Triplets)
2. The Method:
a. Ribosomes bind to nitrocellulose, but mRNA and tRNA do not.
b. Charge Ribosomes with single codon (triplet) mRNA and bind them to the
nitrocellulose.
c. Add the radio-labeled amino acids that have charged their tRNA.
d. Combine the labeled-charged tRNA with the nitrocellulose. If the radioactivity
remains, the codon-anticodon have matched.
e. Analyze the sequence on the codon and anti-codon.
E. The Use of Repeating Copolymers
1. Gobind Khorana - chemically synthesize di-, tri-, and tetra- nucleotides.
2. Dinucleotide: 2 repeating triplets
Trinucleotide: 3 repeating triplets
Tetranucleotide: 4 repeating triplets
3. Technique confirmed other's results and gave light to new assignments.
Including stop codons.
V. The Coding Dictionary
A. Degeneracy and Wobble (Crick, 1966)
1. Third position is usually the variable position
2. Wobble allows the anticodon of a single tRNA species to pair with more than
one triplet in mRNA
a. Can be done without changing the amino acid sequence
b. U may pair with A or G. G may pair with C or U. Inosine may pair with C,U,
or A
c. With this in mind, only 30 different tRNA species are needed to
accommodate the 61 triplets specifying amino acids.
B. Initiation and Termination
1. Bacteria: fmet (N-formylmethionine) with AUG (sometimes GUG)
a. The formyl may be removed or the whole residue removed after synthesis of
the protein.
b. Within the protein, only met added with AUG codon
2. Eukaryotes: AUG start codon adds only met.
3. Termination: UAA, UAG, UGA
a. Nonsense mutation - premature termination caused by an internal amino
acid codon being changed to a stop codon.
amber (UAG)
ochre (UAA)
opal (UGA)
b. Missense mutation - when stop codon is changed to an amino acid codon.
VI. Confirmation of Code Studies: Phage MS2
VII. Universality of the Code
A. Between 1960-1978 the universality of the code was established.
Virtually the same in viruses, bacteria, and eukaryotes.
B. Some exceptions were the yeast and human mitochondria
1. Most variations in wobble position
2. Led to a reduction in the number of tRNA species needed (22)
VIII. Protein Synthesis: Players
A. DNA template
1. Sense and Antisense strands: Initial template binding occurs by the sigma subunit
of the RNA Polymerase enzyme at the promoter sequences of DNA.
a. RNA Polymerase enzyme explores the DNA "looking" for the start points
b. RNA Polymerase is a large enzyme - covers 60 nucleotide pairs
2. Promoters: consensus sequences
(a) -10 (David) Pribnow box 3'TATAATG5' important in binding RNA polymerase
(b) -35 Recognition 3'TTGACA5' distance is important
3. Information codes for: mRNA, tRNA, rRNA
B. RNA Polymerase (1959 - Samuel Weiss)
1. makes all three RNA's
2. Structure
(a) Core = 2a, b, b', sigma polypeptides
(b) Functional subunits
beta polypeptide = provides the catalytic basis and active site
sigma subunit = plays a regulatory function involving the initiation of RNA
transcription
3. Prokaryotes have a single form. Eukaryotes have 3 RNA polymerases
(a) RNA pol I - rRNA, found in the Nucleolus
(b) RNA pol II - mRNA, found in the Nucleoplasm
(c) RNA pol III - 5sRNA and tRNAs, found in the Nucleoplasm
IX. Process
A. Binding
1. RNA Polymerase binds to the promoter regions (-35 and -10)
(a) RNA Polymerase is a large enzyme
(b) relationship between promoters and RNA polymerase may aid in orienting
molecule correctly.
(c) requires the sigma unit (different sigma units control the amount of RNA
transcription)
2. Hydrogen bonds broken (easily at the High A=T regions)
B. Initiation
1. Begins reading the DNA 3' => 5' on the sense strand
2. Approximately 6-7 bases after the -10 (Pribnow Box), first two complements
linked (ribonucleotide triphosphate).
3. Form a DNA-RNA hybrid helix (temporary)
C. Elongation
1. Creates an RNA strand (5' => 3') by adding ribonucleotides to the free 3'-OH
group at a rate of 50 bases/sec (37oC).
2. After about 10 bases, the sigma unit drops off, not required for elongation
D. Termination
1. Palindromic sequences
2. How might they work? rho dependency
a. Rho independent: a long palindrome codes for a large hair-pin, which,
when formed, dissociates polymerase from template.
b. Rho dependent: shorter palindromes create smaller hairpins - a rho subunit is
required to disengage polymerase.
X. Translation: Players
A. mRNA GENE TRANSCRIPT
(a) cloverleaf shape, about 75 to 90 nucleotides - synthesized as a larger
transcript
(b) many post transcriptionally modified bases:
inosinic acid
hypoxanthine
ribothymidylic acid
pseudouridine
(c) TYC arm, pseudouridine arm, amino acid binding arm (3'-CCA), and
anticodon arm, extra arm varies in length (short and long groups)
C. Ribosomes [Svedberg 1S = 10-13s]
(a) synthesized in the nucleolus of the eukaryotic nucleolus. In prokaryotes,
simply synthesized from DNA in cytoplasm.
(b) Prokaryote Eukaryote
70S = 50S and 30S 80S = 60S and 40S
50S = 23S and 5S with 31 proteins 60S = 28S, 5.8S, 5S, and 50 proteins
30S = 16S with 21 proteins 40S = 18S and 33 proteins
(c) rRNA acts as a scaffold to hold the proteins. The 3' end of the 16S rRNA is
important in recognition of the mRNA.
(d) The proteins act as "factors" - initiation, elongation, and termination
(e) E. coli: 7 copies of a single sequence codes for all three rRNA components.
Initial transcript is a 30S RNA that is cleaved into 23S, 16S, and 5S.
(f) Eukaryotes: multiple tandem repeats separated by spacer DNA. Large
transcripts that are cleaved to make the smaller subunits. Example: Humans
45S transcript found on the ends of chromosomes 13, 14, 15, 21, and 22. 5S not
part of this larger transcript (Humans: chromosome 1).
XI. Preparation for Translation: Charging of tRNA
A. An amino acid reacts with ATP by the action of aminoacyl-synthetase to
form Amino Acid-AMP + (PPi). The aminoacyl-synthetase then binds this
activated amino acid to the proper tRNA with the elimination of AMP.
B. Binding to the tRNA is on the 3' end CCA. The adenine connects through the
3'-OH to the carboxyl group of the amino acid.
C. 30 different tRNAs and 20 different aminoacyl-synthetases. Due to the wobble
of the 3rd position in the Codon.
D. "Second Genetic Code" - the specific binding of tRNA to the appropriate amino
acid
XII. Process (Prokaryotes)
A. Initiation
1. Small ribosomal subunit (30S) binds S1 protein to activate the small subunit.
This covers an area of 35-40 bp on the mRNA.
2. The activated small subunit then binds to a consensus sequence at a short
sequence that is complementary to part of a hexamer that lies close to the 3'
end of the 16S rRNA. 5'...AGGAGG...3' called Shine-Dalgarno sequence.
Lies about 7 bases downstream from the AUG start codon.
3. Activated small subunit reads down the mRNA until the start codon AUG is
found. Places the AUG codon in the P site (Peptidyl site).
4. A special tRNA carrying fMet binds to this structure, forming the initiation
complex.
B. Elongation
1. Large subunit "sits" on initiation complex such that the "P" site is occupied by
the fMet-tRNA.
2. The second tRNA, dictated by 2nd codon
3. Translocation reaction
a. peptidyl transferase, an enzyme in large subunit, forms peptide bond
between amino acids.
b. At same time, bond linking the mRNA to tRNA is broken, and the tRNA is
released.
c. Structures (ribosome) moves 3 bases to the right.
d. Requires several elongation factors (EFs)
C. Termination
1. UGA, UAG, UAA => no tRNA's
2. The polypeptide is cleaved from last tRNA by releasing factors. GTP-
dependent release factors: cleave the polypeptide chain from the terminal
tRNA
D. Polyribosomes: repetition of protein synthesis on one mRNA
XIII. Protein Synthesis in Eukaryotes
A. Transcription and Translation in Eukaryotes: Differences from Prokaryotes
1. Transcription in eukaryotes occurs within the nucleus under the direction of 3
separate forms of RNA polymerases; for the mRNA to be translated, it must
move out into the cytoplasm.
2. The initiation and regulation of transcription are under the control of extensive
nucleotide sequences found in DNA upstream from the pint of initial
transcription. Promoters and Enhancers
3. Translation occurs on ribosomes that are larger and whose rRNA and proteins
are more complex than those present in prokaryotes.
4. Protein factors similar to those in prokaryotes guide initiation, elongation, and
termination of translation in eukaryotes. However, there appear to be more
factors required during each of these steps.
5. Initiation of eukaryotic translation does not require the amino acid
formylmethionine. however, the AUG triplet is essential to the formation of
the translational complex and a unique transfer RNA (tRNAimet) is used for
its initiation.
6. Extensive modifications occur to eukaryotic RNA transcripts that eventually
serve as mRNAs. The initial transcripts are much larger than those that are
eventually translated. Thus, they are called pre-mRNAs and are thought to
constitute a group of molecules found only in the nucleus - a group referred to
generally as heterogeneous RNA (hnRNA). Only about 25% of hnRNA
molecules are converted to mRNA. Those that have substantial amounts of
their ribonucleotide sequence excised, while the remaining segments are
spliced back together prior to translation. This phenomenon has given rise to
the concept of so-called split genes in eukaryotes.
7. Prior to the processing of an mRNA transcript, a cap and tail are added to the
molecule. these modifications are essential to efficient processing and
subsequently to translation.
8. Eukaryotic mRNAs are much longer-lived than their bacterial counterparts.
Most exist for hours, rather than minutes, prior to their degradation in the cell.
B. TRANSCRIPTION: Players
1. RNA Polymerase - no subunits known
a. RNA Polymerase I - rRNA
b. RNA Polymerase II - mRNA
c. RNA Polymerase III - tRNA
2. DNA Double Helix
a. higher levels of structure -bending, coiling, etc. (Helicase, Gyrase)
b. Promoter Sequences - Polymerase Specific
(1) RNA pol I - only codes for rRNA genes (multiple copies, but all identical).
Some consensus sequences -38 ATCTTT. But typically, most species
have unique sequences. Easy for RNA Pol I to evolve with changes in
promoter. Similarity increase as ancestry becomes more recent.
(2) RNA Pol II - codes for all different mRNA's. Much more restrictive, can't
evolve with one sequence or lose capacity to transcribe others. As a
result, much greater conservation over evolutionary time. Consensus
sequence among eukaryotes.
-25 TAT[]A[] (Goldberg-Hogness or TATA Box[analogous to -10 in prokaryotes]
-75 GGCCAATCT (CAAT Box)
-110 GC Box
Promoter not required for transcription, but is required for orderly,
repeatable transcription, always starts in same place.
(3). RNA Poly III - tRNA short sequences with internal promoters.
c. Enhancer sequences (discovered 1980): Possibly short sequences involved
in binding transcription factors
(1) regions important to productivity levels, mutation causes 100X decrease in
productivity
(2) Position not fixed relative to gene they enhance. may be up-stream,
down-stream, or in the gene. Can be up to 3000 bp away.
(3) Not gene specific, If the enhancer or the gene is moved, an unrelated
gene will be enhanced.
(4) Sequences may induce formation of Z-DNA, altering degree of
supercoiling, or protein product may aid RNA Pol binding.
3. Transcription Factors - additional proteins required for transcription. Aid in
RNA pol lII binding to promoter
a. TATA - factor (TFIID): Binds to promoter
b. also factors for GC region and CCAAT Box
c. supplant role of sigma factor in prokaryotes. Greater degree of specific control
C. Process
1. Mechanics (similar to prokaryotes)
a. Binding of RNA Pol to promoter; efficiency modified by enhancers and
transcription factors.
b. Transcribes by linking complementary ribonucleotides
c. Termination - similar (palindromic regions)
2. Modifications
a. 1970, James Darnell, recognized that initial mRNA products are considerably
longer than cytoplasmic mRNA that is translated.
1. Heterogeneous RNA (hnRNA). Found only in nucleus, not in the cytoplasm
2 By 1977, Susan Berget and Philip Sharp found that there were internal
sequences in hnRNA that were not present in the mRNA. Regions spliced out
in mRNA may be 20,000 bp long (introns), and intervening sequences spliced
together (exons). Splicing accomplished by Spliceosome and snRNA (small
nuclear ribonucleoproteins). Made-up of an RNA scaffold with SNURPS.
[40S yeast, 60S mammals] Obviously, implies that intervening sequences
exist between coding regions in the DNA. DNA => Intervening Sequences -
Introns (interruptions) => coding Sequences - Exons (expressed)
c. Recognition of the Exon Regions
(1) Sequence analysis of RNA product in relation to the DNA sequence
showed portions of the DNA sequence were missing in the mRNA of the
cytoplasm
(2) Formation of heteroduplexes: Bind DNA to RNA, it will bind where
complementary and antisense will have to "loop-out". The "loop-out"
regions were the introns
(3) Splicing Mechanisms vary depending on the RNA being produced
tRNA: Endonuclease and Ligase
rRNA: Self splicing, Ribozyme
mRNA: Lariat Formation
Mitochondrial RNA: RNA maturase
d. Mechanics of splicing
(1) Each RNA is spliced in a different way
(2) Splicing is catalyzed by a series of endonucleases and ligases coalesced
into a large enzymatic unit called the spliceosome.
(3) Tom Maniatis (1991) The concentration of endonucleases and associated
splicing factors influences the position of the cut. If SXL absent - cut is
made at site 1, and UAG preemptively stops translation, no functional
protein produced. If SXL splicing protein is present, it binds to site 1 and
cut is made at 2-. Functional protein produced.
3. Review
a. Each RNA Polymerase recognizes its own promoter
b. To produce mRNA, RNA pol II recognizes the TATA Box (-25) and/or CAAT
Box (-75), and transcribes to terminator (introns and exons)
c. Introns spliced out - may be tissue specific regulation based on concentration
of splicing proteins in spliceosome.
d. 5'-Cap and 3'-polyA tail added.
e. mRNA transported to the cytoplasm
C. Translation : Players:
1. mRNA, with start and stop codons
2. Ribosome: 80S => large subunit 60S, small subunit 40S
Large subunit = 28S, 5.8S, and 5S rRNA + 50 proteins
Small subunit = 18S + 33 proteins
3. tRNA - same specificity
D. Process
1. Mechanics: same as prokaryotes EXCEPT: mRNA binds to small subunit after
the tRNAimet. The mRNA then binds and the whole unit moves down the
mRNA to the start codon where the large subunit binds.
2. Modifications: Post translational modifications
a. Adding sugar, lipid, or protein groups (not exclusive to eukaryotes)
b. Splicing (Patricia Kane 1990) : Yeast TFP1 gene - codes for 1 translational
product which is cleaved into 2 functional proteins.
E. Exceptions:
1. Non-universality: some triplet codons have different results in different
organisms
a. UGA - usually a terminator, but in human mitochondria and yeast
mitochondria it codes for tryptophan
b. UAG - usually a terminator, but in paramecium it codes for glycine.
c. five other exceptions are known. Really not many, given the extensive work
done on a variety of organisms.
2. Overlapping Genes
a. fX174 virus has circular chromosome consisting of 5386 nucleotides that
could encode a maximum of 1795 amino acids, enough for 5 to 6 proteins.
But, it produces 11, consisting of over 2300 amino acids.
Prokaryote Transcription
I. Operons
A. For greatest efficiency, the genes for proteins that function together are
controlled together. Coordinate control through grouping functionally related
genes together so they can be regulated together.
B. The lac Operon
1. Lactose metabolism in E. coli is carried out by two enzymes, with possible
involvement by a third.
a. galactose permease = lacY transports lactose into cells
b. beta galactosidase = lacZ cuts the beta-galactosidic bond between galactose and
glucose
c. galactose transacetylase = lacA function in lactose metabolism is unclear
2. The genes for all three enzymes are clustered together and transcribed
together from one promoter, yielding a polycistronic message.
a. cistron is a synonym for gene
3. Therefore, these three genes, linked in function, are also linked in expression.
4. They are turned off and on together
C. Negative Control of the lac Operon: Occurs as follows:
1. Negative control implies that the operon is turned on unless something
intervenes to stop it
2. The operon is turned off as long as repressor binds to the operator, because
the repressor interferes with RNA polymerase's binding to the adjacent
promoter.
a. lac repressor: product of the lacI gene is a tetramer of four identical protein
chains that bind to the operator
b. When the repressor is bound to the operator, the operon is repressed
c. Operator and promoter are contiguous
d. If RNA Polymerase cannot transcribe the lacZ, T, and A genes, the operon is
off, or repressed.
i. Structural Genes: lacZ, lacY, and lacA
ii. Regulatory Genes: Promoter and Operator
3. When the supply of glucose is exhausted and lactose is available, the few
molecules of lac operon enzymes produce a few molecules of allolactose
from the lactose.
a. Lac operon is repressed as long as glucose is present
b. Repressor: allosteric protein
c. Changes its conformation, therefore its function when it binds to and inducer
molecule
d. Allolactose: when beta-galactosidase cleaves lactose to galactose plus
glucose, it rearranges a small fraction of the lactose to allolactose.
i. Allolactose is just galactose linked to glucose in a different way than in
lactose.
ii. In lactose, the linkage is through a beta-1,4 bond; in allolactose, the linkage is
beta-1,6.
4. Allolactose acts as an inducer by binding to the repressor and causing a
conformational shift that encourages dissociation from the operator.
5. With the repressor removed, RNA polymerase is free to bind to the lac
promoter and transcribe the three structural genes.
D. Operon Hypothesis
1. Francois Jacob and Jacques Monod (1940-50) Pasteur Institute
2. By 1950 realized that the three enzymes were induced together
a. Constitutive mutants: that needed no induction
b. They produced the three gene products all the time
3. Arthur Pardee, Jacob and Monod created merodiploids (partial diploids)
carrying both the wild-type (indelible) and constitutive alleles of the lac
operon.
a. Indelible allele proved to beta dominant, demonstrated that wild-type cells
produced some substance that turned the lac genes off
b. lac repressor: controlled both wild-type and merodiploids
4. Existence of a repressor required that there be some specific receptor to
which the repressor would bind. Jacob and Monod called this the Operator.
a. cis-dominant: it is dominant only with respect to genes on the same piece
of DNA (in cis; Latin: cis = here) mutations are called Oc, for operator
constitutive.
b. mutations in the repressor gene: dominant both in cis (on same
chromosome) and in trans ( on different chromosome) because the mutant
repressor will remain bound to both operators even in the presence of the
inducer.
c. Suzanne Bourgeois: later found many other mutants. These are named Is to
distinguish them from constitutive repressor mutants (I-) which make a
repressor that cannot recognize the operator.
E. Positive Control:
1. It would be wasteful for E. coli to turn on the lac operon in the presence of
glucose. E. coli cells keep the lac operon turned off as long as glucose is
present. Geneticists suspected that this selection in factor of glucose
metabolism and against use of other energy sources was due to the
influence of some breakdown product, or catabolite. Hence the name
Catabolite Repression
2. Positive control (catabolite repression) of the lac operon works as follows: a
complex composed of cAMP plus a protein known as catabolite activator
protein (CAP) binds to the upstream part of the promoter and facilitates
binding of RNA polymerase to the down stream part.
a. One substance that responds to glucose concentration is a nucleotide called
cyclic AMP (cAMP). The lower the level of glucose, the higher the
concentration of cAMP.
b. Positive controller of the lac operon is a complex composed to two parts:
cAMP and a binding protein called CAP, for Catabolite activator protein by its
discoverers, Geoffrey Zubay and his colleagues. The protein binds cAMP,
and the resulting complex binds to the lac promoter and turns it on by
helping RNA polymerase bind. Notice that the lac promoter is divided into
two parts, the CAP binding site on the left and the RNA polymerase binding
site on the right.
c. CAP and cAMP also stimulate transcription of other inducible operons,
including the well-studied ara and gal operons.
3. This greatly enhances transcription of the operon.
4. The physiological significance of this positive control mechanism is that it can
only operate when cAMP concentration is elevated; this occurs when
glucose concentration is low and there is a corresponding need to use an
alternate energy source.
II. Temporal Control of Transcription (Viral early and late genes. Bacillus subtilis
sporulation genes)
A. Modification of the Host RNA Polymerase
1. Bacillus subtilis: and phage SPO1
2. SPO1: large phage with many genes
a. Transcription of phage SPO1 genes in infected B. subtilis cells proceeds
according to a temporal program in which early genes are transcribed first,
then middle genes, and finally late genes.
i. first five minutes: early genes are expressed
ii. middle genes turn on about 5 to 10 minutes post-infection
iii. from about the 10 minute point until the end of infection, the late genes
switch on
b. Switching is directed by a set of phage-encoded sigma factors that associate with
the host core RNA polymerase and change its specificity from early to
middle to late.
i. phage does not carry its own RNA polymerase
ii. B. subtilis holoenzyme: 2alpha, beta, beta'
iii. sigma factor MW= 43,000
iv. one of the genes transcribed in the early phage of SPO1 is called gene 28
gp28 (gene product) displaces host sigma (sigma43)
gp28 changes the RNA pol specificity: transcribes the phage middle
genes
gp28 is a novel sigma factor that accomplishes two things;
(1) it diverts the host's polymerase from transcribing host genes
(2) it switches from early to middle transcription.
c. Host sigma is specific for the page early genes; the phage gp28 witches the
specificity to the middle gene; and the phage gp33 and gp34 switch to late
specificity.
i. gp33 and gp34 products of two phage middle genes replace gp28
ii. Note that the polypeptides of the host core polymerase remain constant
throughout this process; it is the progressive substitution of sigma factors that
changes the specificity of the enzyme and thereby directs the
transcriptional program.
B. RNA Polymerase encoded in Phage T7
1. T7: smaller genome than SPO1
a. distinguish three phases of transcription: class I, class II, and class III
2. Phage T7, instead of coding for a new sigma factor to change the host
polymerase's specificity from early to late, encodes a whole new RNA
polymerase with absolute specificity for the later phage genes.
3. This polymerase, composed of a single polypeptide, is a product of one of the
earliest phage genes, gene 1.
4. The temporal program in the infection by this phage is simple. The host
polymerase reads the earliest (class I) genes, one of whose products is the
phage polymerase, which then reads the later (class II and class III) genes.
C. Control of Transcription During Sporulation
1. Growth state: vegetative. Onset of hard times forms protective endospores
through sporulation
2. When the bacterium B. subtilis sporulates, a whole new set of sporulation-
specific genes turns on, and many, but not all vegetative genes turn off.
3. Switch takes place largely at the transcription level.
4. Accomplished by several new sigma factors that displace the vegetative sigma factor
from the core RNA polymerase and direct transcription f sporulation genes
instead of vegetative genes.
i. sigma29, sigma30, and sigma32 (sigmaE, sigmaH, and sigmaC) for sporulation
ii. sigma43 (sigmaA) for vegetative
5. Each sigma factor has its own preferred promoter sequence.
6. E. coli also has multiple sigma factor with their own specificities. Heat-shock
genes sigma32 (or sigmaH)
D. Infection of E. coli By Phage l
1. Phage l can replicate in either of two ways: lytic or lysogenic.
2. In the lytic mode, almost all of the phage genes are transcribed and
translated, and the phage DNA is replicated, leading to production of progeny
phages and lysis of the host cells.
3. In the lysogenic mode, the l DNA is incorporated into the host genome; after
that occurs, only one gene is expressed.
4. The product of this gene, the l repressor, binds to the two early phage
operators and prevents transcription of all the rest of the phage gene.
5. However, the incorporated phage DNA ( the prophage) still replicates, since it
has become part of the host DNA.
6. Lytic Replication of Phage l
a. Linear, as the DNA exists in the phage particles, and circular, the shape the
DNA assumes shortly after infection begins.
i. 12-base overhangs,, or sticky ends
ii. cohesive ends go by the name cos
iii. cyclization brings together all the late genes, which had been separated at
the two ends of the linear genome.
b. The immediate early/delayed early/late transcriptional switching in the lytic
cycle of phage l is controlled by antiterminators.
i. host RNA polymerase holoenzyme transcribes the immediate early genes
first. cro and N
ii. One of the two immediate early genes is cro, which codes for an
antirepressor that allows the lytic cycle to continue.
iii. The other, N, codes for an antiterminator, pN, that over rides the
terminators after the N and cro genes.
iv. cro and N lie immediately downstream from the rightward and leftward
promoters, PR and PL
v. At this stage no repressor is bound to the operators that govern these
promoters (OR and OL): transcription proceeds unimpeded.
vi. When polymerase reaches the ends of the immediate early genes, it
encounters. r- dependent terminators and stops short of the delayed early
genes.
vii. Same promoters (PR and PL) are used for both immediate early and
delayed early transcription. Does not involve a new sigma factor or RNA
polymerase. it involves an extension of transcripts controlled by the same
promoters.
c. Transcription then continues on into the delayed early genes. establishes
lysogeny.
i. rightward side, downstream from cII, genes O and P code for protein
necessary for phage DNA replication.
ii. Q gene product (pQ) = antiterminator which permits transcription of late
genes.
iii. One of the delayed early genes, Q, codes for another antiterminator (pQ)
that permits transcription of the late genes from the late promoter, PR', to
continue without premature termination.
d. Late genes are all transcribed in the rightward direction
i. late promoter PR' lies just downstream from Q
ii. Transcription from this promoter terminates after only 194 bases, unless pQ
intervenes to prevent termination.
iii. late genes code for the proteins that make up that phage head and tail, and
for protein that lyse the host cell so the progeny phage can escape.
7 Establishing Lysogeny
a. delayed early genes are required for lytic and lysogenic cycles
i. most delayed early gene products are needed for integration of the phage
DNA into the host genome
ii. products of the cII and cIII genes allow transcription of the cI gene and
therefore production of the l repressor , the central component in lysogeny.
b. Phage l establishes lysogeny by causing production of enough repressor to
bind to the early operators and prevent further early RNA synthesis
c. Promoter used for establishment of lysogeny is PRE, which lies to the right of
PR and cro.
i. RE in PRE stand for repressor establishment.
ii. PRE lies to the right of both PR and cro.
iii. It directs transcription leftward through cro and then through cI
d. Transcription form this promoter goes leftward through the cI gene.
e. another promoter, PRM, comes into play later, to maintain lysogeny.
i. RM in PRM stand for repressor maintenance. promoter used during lysogeny
to ensure a continuing supply of repressor to maintain the lysogenic state.
ii. Peculiar property of requiring own product (repressor) for activity.
iii. this promoter cannot be used to establish lysogeny because there is no
repressor to activate it at the beginning of infection
8. Lysogen Induction:
a. E. coli cells respond to environmental insults, such as mutagens or radiation,
by inducing a set of genes whose collective activity is called the SOS
response
i. When a lysogen suffers DNA damage, it induces the SOS response.
ii. recA product participates in recombination repair of DNA damage but
environmental insults also induce a new activity in the recA protein -
stimulates a latent protease, or protein-cleaving activity in the l repressor.
c. The initial event in this response is the appearance of co-protease activity in
the RecA protein.
d. This causes the l repressors to cut themselves in half, removing them from
the l operators and inducing the lytic cycle.
d. In this way, l phages can escape the potentially lethal damage that is
occurring in their host.
III. Specific DNA-Protein Interactions
A. Proteins that bind to specific regions of DNA
1. l repressor and cro bind to their respective operators
2. CAP which binds to the CAP binding site in the lac promoter
B. They can locate and bind to one particular short DNA sequence among a vast
excess of unrelated DNA
C. All the proteins just listed have a similar structural motif: two alpha helices
connected by a short protein turn {helix-turn- helix}