Last revised: Monday, March 10, 2003
Reading: Ch. 13 in text
Regulation of gene expression in prokaryotes
Many genes occur in operons
- several structural genes (for protein) controlled by a single promoter
- mRNA is polycistronic: has multiple start & stop signals, codes for multiple polypeptides
- In some cases, transcription depends on specific DNA region, the operator, which is near or overlapping the promoter.
- Operator site can bind repressor proteins, controlled by some effector molecule
Transcription & Translation are coupled
- Prot. synthesis begins before transcription ends
- multiple ribosomes bind to each mRNA
- All control at level of transcription
- once m-RNA is made, it is translated.
Some genes are expressed constitutively
- Always translated when cells synthesizing protein
- Examples
- ribosomal genes
- genes for replication & transcription
- genes for central metabolism enzymes
- Note: some proteins made from constitutive genes occur at much higher levels than others. Why? Strong vs. weak promoters.
Some genes are regulated by repressor proteins
- Classic example: the lac operon
- gene is regulated by negative control; in absence of specific repressor, gene is transcribed just like constitutive gene. In order to regulate, must add specific block. Must say "no"; otherwise gene is not down-regulated.
- View Animation of negative control from Cornell University
- Lactose = milk sugar, disaccharide made of galactose + glucose. In order to metabolize lactose, cells must produce enzyme ß-galactosidase, split lactose into galactose + glucose
- Observation: add lactose to cells: within minutes, ß-galactosidase enzyme appears, also lac permease in membrane, and a third protein, transacetylase. Level of ß-galactosidase enzyme can accumulate to level of 10% of cytoplasmic protein!
- Explanation: in absence of lactose (= inducer), lac repressor blocked operator site.
- Lac repressor is allosteric protein. Coded for by another region of DNA (constitutive gene, weak promoter, low level of expression)
- View diagram showing binding of repressor protein to DNA
- Effector molecule = Inducer is lactose (actually allolactose, or analog such as IPTG) binds to repressor protein, repressor released, RNA is made, all genes turned on as unit
- View animation of lac operon (access to Campbell Biology 6th Ed. website required)
- View animated movie of "The Lactose Paradigm"
- In lab, MaConkey agar used to detect lac mutants. If lactose is used (lac genes on), fermentation occurs in colony, acid produced, color indicator turns pink. Pink colony = lactose utilized. But if lactose genes don't work (mutant), colonies grow but stay white.
- To review these concepts, look at Cornell University's negative control explanation.
Some genes are regulated by activator proteins
- genes with weak promoters are rarely transcribed
- some such genes can be activated by an activator protein, causes RNA polymerase to bind more tightly.
- often there are two components to such regulation: a sensor protein and an activator protein. The activator protein is inactive until phosphorylated by the sensor, then it activates transcription, gene product is made.
- See animation of positive control.
- See animation of catabolite repression
Quorum sensing
- Some genes are only turned on when bacteria are present in high concentrations.
- Q. How can bacteria "know" that other bacteria of their species are present in abundance?
- A. Many bacteria can use "quorum sensing" to detect presence of specific autoinducer chemicals used for measuring cell density. These autoinducers are specific peptides (gram positive bacteria) or homoserine lactones (gram-negative bacteria). See structure of some autoinducers. ("protected" image.) Why is this protected?
- Autoinducer is produced in low amounts by all cells, diffuses freely across membrane, so concentration inside and outside cell is same. As cell number increases, autoinducer conc. increases until it is sufficient to bind to activator, turn on transcription of specific genes. See mechanism of quorum sensing. ("protected" image.) Why is this protected?
- Example: some virulence genes of pathogens are controlled by quorum sensing. Until a sufficient density of cells is present, cells don't turn on these factors.
Protein synthesis (in prokaryotes)
- carries coding information for amino acids = codons, 3 adajacent nucleotide bases
- Example: AAA, AGU, etc.
- leader sequence on mRNA (called Shine-Dalgarno sequence) binds to complementary sequence on small ribosome subunit.
- View components of ribosomes
- acts as a "decoding box" or "tape player" for the information in mRNA
- 30S & 50S subunits (= 70S)
- 30S has 16S RNA + 21 proteins
- 50S has 23S & 5S RNA + 34 proteins
- View model of ribosome
- View model of ribosome
- structure: 4 loops, anticodon, AA binding site
- ~ 60 types in bacteria (>100 in mammals)
- only 73-93 nucleotides long
- some modified bases: pseudouridine, inosine, others
- modified after transcription
- extensive hairpin loops
- View model of tRNA
- anticodon site: recognizes codon on mRNA
- AA added by enzyme: AA-tRNA activating enzymes
- ATP required, forms AA-AMP + PP, then AA-tRNA + AMP
- View static model of translation
- View animation of translation #1
- View animation of translation #2
- Interact with shockwave interactive simulation of translation
- Initiation
- 30S initiates binding to mRNA
- locates Shine-Dalgarno sequence (3-9 bases near 5' end of mRNA)
- ribosome finds first AUG codon
- 50S ribosome binds
- tRNA carries N-formylmethione to first position
- View animation of initiation (access to Campbell Biology 6th Ed. website required)
- Elongation
- 2 adjacent sites on ribosome: P and A site
- A site accepts a new tRNA-AA
- Psite holds existing chain
- peptide transferred from P site tRNA to A-site AA
- enzyme activity is in ribosomal RNA, not protein
- also required: Energy (GTP) and elongation factors
- View animation of elongation (access to Campbell Biology 6th Ed. website required)
- Termination
- reach a "stop codon" UAG, UAA, or UGA
- no t-RNAs for release, but release factors required
- View animation of termination (access to Campbell Biology 6th Ed. website required)
- Initiation
- Net cost: 4 phosphate bonds/amino acid added!
- AUG = universal "start" codon
- UAG, UAA, UGA = "stop" codons
- A few messages in bacteria use GUG as start, but still need Shine-Dalgarno sequence, still code for N-formylmethionine
- Genetic Code table (arranged to view codons for each amino acid)
GCA
GCC
GCG
GCU
AlaAGA
AGG
CGA
CGC
CGG
CGU
Arg
GAC
GAU
Asp
AAC
AAU
Asn
UGC
UGU
Cys
GAA
GAG
Glu
CAA
CAG
Gln
GGA
GGC
GGG
GGU
Gly
CAC
CAU
His
AUA
AUC
AUU
IleUUA
UUG
CUA
CUC
CUG
CUU
Leu
AAA
AAG
Lys
AUG
Met
UUC
UUU
Phe
CCA
CCC
CCG
CCU
ProAGC
AGU
UCA
UCC
UCG
UCU
Ser
ACA
ACC
ACG
ACU
Thr
UGG
Trp
UAC
UAU
Tyr
GUA
GUC
GUG
GUU
Val
- when looking at DNA sequences, see many AUG (approx. 1 in every 64). But most of these are not actual "start" codons. Why?
- 3 possible reading frames. Also, AUG serves as ordinary codon for methionine.
- Most proteins are long (100s to thousands of bases). Look for AUG followed by long region without "stop" codon = ORF
- Computer programs used to find ORFs from DNA sequences
- ORF = gene, defined by computer search
- originally thought all organisms use identical codons
- But mitochondria of eukaryotes (except plants) use slightly different assignments for a few codons. Some examples of non-universal codons are shown in the table below.
Codon"Universal"
CodeMammalian
Mitochondrial CodeYeast
Mitochondrial CodeUGA Stop Trp Trp AUA Ile Met Met CUA Leu Leu Thr AGA
AGGArg Stop Arg
- original assumption: if multiple codons for an amino acid, expect equal frequency of use
- Surprise! Codon use is often highly biased. Eg. E. coli can use AUA, AUU, or AUC to specify isoleucine; but only 1 in 20 times is an isoleucine coded by AUA; 19/20 times encoded by AUU or AUC. So AUA is only rarely used -- may allow evolution to develop alternate codes.
- Consult the codon usage table to find how frequently each codon is used for any organism in this extensive database.