Download 2054, Chap. 12, page 1 I. Genes: Expression and Regulation A

2054, Chap. 12, page 1
I. Genes: Expression and Regulation
A. DNA transcription or RNA synthesis
1. transcription = synthesis of RNA under direction of DNA
a. RNA has sequence complementary to DNA template
b. uracil replaces thymine
c. mRNA = contains message for protein synthesis
d. tRNA = carries amino acids during protein synthesis
e. rRNA = ribosome components
2. mRNA is single stranded RNA containing directions for protein synthesis
a. leader sequence = nontranslated sequence at 5' end preceding initiation codon
b. polygenic mRNAs (code for more than one polypeptide) have spacer regions
separating segments for individual polypeptides
c. trailer = nontranslated sequence at 3' end, after the last termination codon
3. RNA polymerase synthesizes mRNA
a. requires NTP, DNA template
b. 5' ÷ 3'
c. copies only the sense strand of DNA
(1) different genes may be encoded on opposite strands
(2) gene = DNA segment or sequence that codes for a polypeptide, an rRNA, or a
tRNA
4. RNA polymerase (E. coli)
a. opens double helix and transcribes the sense strand to produce RNA transcript
that is complementary and antiparallel to DNA template
b. large molecule with 4 polypeptides (", $, $', F)
(1) core enzyme = "2, $, $' = catalyzes RNA synthesis
(2) sigma factor = no catalytic activity, helps core enzyme recognize the promoter
(3) rifampin binds to the $ subunit
5. promoter = 6 base sequence, about 35 base pairs before transcription start point
a. usually TATAGACA
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b. Pribnow box (TATAAT) about 10 bases upstream of transcript
c. RNA pol recognizes, binds to promoter, and unwinds short DNA segment around
Pribnow box
6. terminators = stop transcription
a. all contain sequence that forms a hairpin loop (H2-bonding) that stops or slows
RNA polymerase
b. two types of stop signals or terminators
(1) UUUUUU after hairpin causes polymerase to stop and release mRNA without
any accessory factors
(2) rho factor (D) (no UUUUUU region)
(a) rho binds to mRNA, moves until it reaches paused RNA polymerase
(b) causes polymerase to dissociate from mRNA, probably by unwinding
mRNA-DNA complex
7. synthesis of rRNA and tRNA similar to mRNA except
a. molecules terminated by 5'-monophosphate rather than triphosphate found at end
of all primary transcripts (mRNA)
b. rRNA and tRNA are smaller than primary transcripts
c. tRNA contains bases other than A, G, C, U not in original transcript
d. primary transcripts undergo posttranscriptional modification or RNA
processing
8. In eucaryotes 3 RNA polymerases instead of 1
a. different promoters
b. heterogeneous nuclear RNA (hnRNA) = large RNA precursors, about 5000 50000 nucleotides long
c. posttranscriptional modification = hnRNA cleaved to final mRNA
d. RNA splicing removes introns from initial RNA transcript
(1) small nuclear RNA (snRNA) binds to splice junctions
(2) splicing of pre-mRNA occurs in large complex called a spliceosome
e. ribozyme = self-splicing pre-rRNA molecules
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f. rRNA and tRNA result from posttranscriptional processing
g. ribozyme = self-splicing
B. Translation
1. mRNA nucleotide sequence is translated into an amino acid sequence
a. amino acids added to growing chains at the carboxyl end (C-terminal)
b. mRNA often complexed with several ribosomes at once, synthesizing several
copies at once (polyribosome)
c. ribosomes can attach to mRNA being synthesized, so transcription and translation
can occur simultaneously (in eucaryotes, transcription and translation machinery
separated by nuclear membrane)
2. amino acid activation occurs during 1st stage of protein synthesis
a. amino acids attached to tRNA molecules at 3' end (CCA) by aminoacyl-tRNA
synthetases
Amino acid + tRNA + ATP ÷ aminoacyl-tRNA + AMP + PPi
b. at least 20 aminoacyl-tRNA synthetases
(1) no proofreading function in protein synthesis
(2) aminoacyl-tRNA synthetases can proofread and will hydrolyze amino acid
from tRNA is incorrect
c. anticodon triplet complementary to mRNA codon
3. Ribosomes are 70S organelles composed of a 50S and 30S subunits
a. each subunit constructed of 1 or 2 rRNA molecules and many proteins
b. both subunits contribute to the translational domain, the region directly
responsible for translation
c. eucaryote ribosomes are 80S (60S + 40S)
4. Protein synthesis can be divided into initiation, elongation, and termination
5. most bacteria begin protein synthesis with a specially modified aminoacyl-tRNA, Nformylmethionyl-tRNAfmet
a. amino acid blocked by formyl group
b. used only for initiation
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c. eucaryotes and archaebacteria begin with methionyl-tRNAmet
d. formyl group is hydrolyzed; 1-3 terminal amino acids may also be removed
e. initiation factors (3 in procaryotes) are required for proper binding and
arrangement of the complex
6. elongation divided into aminoacyl-tRNA binding, transpeptidation, and translocation
a. aided by elongation factors
b. two sites for binding aminoacyl-tRNA and peptidyl-tRNA
7. at beginning of elongation, N-formylmethionyl-tRNAfmet or peptidyl-tRNA is in the
peptidyl site (aminoacyl-tRNA site empty at first)
8. the transpeptidation reaction catalyzed by peptidyl transferase
a. initiated when aminoacyl-tRNA binds to the aminoacyl site
b. peptide chain (on peptidyl-tRNA in P site) transfers to the A site tRNA, increasing
length by 1 amino acid at carboxyl end
9. in translocation:
a. peptidyl-tRNA moves from A site to P site
b. ribosome moves one codon along mRNA so that new codon is positioned at the A
site
c. empty tRNA leaves the P site
10. protein synthesis stops when ribosome reaches a nonsense or stop codon
a. release factors aid recognition of these codons
b. once stopped, peptidyl transferase hydrolyzes the peptide free from its tRNA and
the empty tRNA is released
c. ribosome dissociates from mRNA and separates into 30S and 50S subunits
11. as polypeptide leaves the ribosome, it begins folding into its final shape
12. molecular chaparone proteins are proteins that help polypeptides fold into the
proper shape
a. suppress incorrect folding and may reverse incorrect folding
b. often called heat-shock proteins or stress proteins because their concentration
increases when cells are exposed to high temperatures, metabolic poisons, and
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other stressful conditions
(1) help protect cell proteins from denaturing
(2) high concentrations occur normally in hyperthermophiles
13. chaparones also help with protein secretion
C. Regulation is essential for optimizing cell efficiency
1. respond to environment
2. maintain proper levels of cellular constituents
3. goal is to maximize the production and use of energy (cell’s “job” is to make more
cells)
4. carbon flow can be regulated in three ways:
a. localization of metabolites and enzymes = compartmentalization = metabolic
channeling
b. allosteric regulation of specific enzymes
c. transcriptional control (amount of enzyme synthesized)
D. Transcriptional control
1. protein synthesis requires a lot of energy (ATP), so regulation of protein synthesis is
important to cell's energy economy
2. mRNA transcription is regulated by sigma factors, induction/repression, and
attenuation
3. the use of different sigma factors regulates expression of different genes by
controlling the way RNA polymerase binds to the promoter
a. different sigma factors allow RNA polymerase to recognize specific promoters
b. common in G+, G-, and bacteriophages
4. transcription of inducible enzymes can be regulated by induction and repression
a. constitutive = enzymes that are produced continuously
(1) 60 - 80% of genes
(2) usually code for enzymes required needed in fairly constant amounts for major
life processes (e.g., glycolytic enzymes)
b. transcription of an inducible enzyme increases in the presence of an inducer
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c. transcription of repressible enzymes is decreased by corepressors
5. repression = regulatory mechanism that inhibits gene expression and decreases the
synthesis of enzymes (negative regulation)
a. usually a response to an endproduct of a pathway that acts as corepressor
b. leads to decrease in synthesis of enzymes of that pathway that leads to that
product
c. corepressor activates repressors = regulatory proteins that block RNA polymerase
from initiating transcription
6. induction = process that turns on the transcription of a gene or genes
a. inducer = substance that induces transcription by binding to and inactivating a
repressor
b. best known example are the genes for lactose metabolism in E. coli
(1) in absence of lactose, $-galactosidase activity is almost absent ($galactosidase hydrolyzes lactose to glucose and galactose)
(2) in presence of lactose, large amounts of $-galactosidase are synthesized
(3) in cell, lactose converted to allolactose, which induces the genes
(4) so presence of lactose indirectly induces the cells to synthesize more enzyme =
enzyme induction
7. lactose metabolism involves 3 structural gene products (enzymes) located together on
the chromosome along with a control region, all called the lac operon
a. operon = operator + promoter + structural genes
b. structural genes for 3 enzymes are next to each other and regulated together
(1) $-galactosidase
(2) $-galactosidase permease, involved in lactose transport into the cell
(3) thiogalactoside transacetylase, metabolizes certain disaccharides other than
lactose
c. operator = regulatory site that gives a stop or go signal to transcription
d. promoter = region of DNA where RNA polymerase initiates transcription
e. I gene = regulatory gene near the lac operon that codes for a repressor protein
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f. in absence of lactose, repressor protein binds to operator site, preventing
transcription (and expression of structural genes)
g. in presence of lactose, some diffuses into cells and is converted into the inducer
allolactose
(1) inducer binds to repressor protein and alters it (allostery) so that it cannot bind
to the operator site
(2) in absence of bound repressor, structural genes are transcribed into mRNA
(3) transcribed mRNA translated into enzymes
(4) lactose induces enzyme synthesis, lac operon is called an inducible operon
8. lac operon also regulated by catabolite activator protein (CAP) or cyclic AMP
receptor protein (CRP) and the small cyclic nucleotide 3',5'-cyclic adensosine
monophosphate (cAMP) (positive regulation)
a. CAP must bind before to CAP site on promoter before RNA polymerase can
attach and begin transcription (= activator)
b. CAP can only bind when complexed with cAMP
c. cell prefers glucose over lactose because it's more directly metabolized as carbon
and energy source
(1) glycolytic enzymes constitutive
(2) at low levels of glucose, cell makes cAMP
d. cAMP = alarmone, an alarm signal in response to environmental or nutritional
stress (i.e., lack of glucose)
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9. catabolite repression = inhibition of metabolism of alternative carbon sources by
glucose
a. not really repression since it is slow metabolism that stimulates synthesis of the
enzymes
b. also called the glucose effect
E. attenuation = control mechanism that allows transcription to start, but then prematurely
terminates due to secondary structures in mRNA
1. common with amino acid synthesis, trp operon best studied
2. mechanism involves transcription and translation
3. transcription of leader sequence occurs before structural genes
a. in presence of trp, polymerase reaches the attenuator site where transcription
stops
b. in absence of trp, synthesis of gene is normal and all enzymes are made
4. termination is determined by RNA secondary structures
a. two distinctive, mutually exclusive structures
b. structure formed based on rate of translation of the previous region of the leader
sequence into the leader peptide
(1) leader peptide has no cellular function other than regulation
(2) contains two sequential trp codons
(3) in presence of trp, translation continues until a stop codon (UGA)
(a) in this position, the terminator hairpin forms, preventing continued
transcription of the structural genes
(b) polyU region immediately after hairpin
(4) in absence of trp, translation stops earlier and terminator hairpin doesn't form,
so transcription continues through structural genes
F. Global regulatory systems
1. some regulatory systems affect many genes and pathways simultaneously
a. catabolite repression
b. SOS DNA repair
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c. production of heat-shock proteins
d. sporulation
2. several possible mechanisms
a. protein repressor or activator
b. sigma factor
c. nonprotein regulator (e.g., guanosine tetraphosphate)
3. regulon = collection of genes or operons controlled by the same regulatory protein
a. operons usually associated with a single pathway or function
b. e.g., heat-shock proteins, glycerol catabolism
4. modulon = operons controlled by their own regulators that are also under the control
of a common global regulatory protein (e.g., catabolite repression)
5. stimulon = regulatory system in which all operons respond together to an
environmental stimulus in a coordinated fashion
a. may contain several regulons and modulons
b. genes involved in response to phosphate limitation
6. antisense RNA is complementary to another RNA molecule
a. highly specific interaction
b. can block DNA synthesis, mRNA synthesis, or translation
c. genes are called antisense genes
d. can be used to shut down specific genes and observe phenotypic changes
e. may have therapeutic value against pathogens, genetic diseases, or cancer