Bacterial Gene Expression 1

Question 1

Fundamental Issues of Gene Transcription

Answer 1

• what determines the start site of transcription for individual genes
• what is the mechanism of RNA synthesis
• how is the level of transcription of individual genes determined
• how does the level of expression of some genes change

Question 2

Constitutive Gene Expression

Answer 2

-genes expressed throughout the life time of the cell

Question 3

Methods of Specific Protein Levels Control

Answer 3

• Regulation of mRNA synthesis
• -rate of transcription initiation
• -frequency of transcriptional read through
• Regulation of mRNA Degradation
• Regulation of Protein Synthesis - rate of translation initiation
• Regulation of Protein Degradation

Question 4

Transcription

Answer 4

• involves melting the DNA template at the site of transcription, the ‘transcription bubble’
• single stranded DNA is exposed
• RNA polymerase synthesises RNA strands using DNA as template
• complimentary strand to the RNA sequences is the template strand / non-coding strand / antisense strand
• RNA sequence is identical to the coding/sense strand of the DNA except the substitution of Us for Ts

Question 5

Labelling Positions in the DNA Sequence

Answer 5

• DNA nucleotide encoding the beginning of the RNA chain is the transcription start site and is designated the +1 position (there is no 0)
• sequences in the direction in which transcription proceeds are given positive numbers and are referred to as downstream
• sequences preceding the transcription site are upstream sequences and their positions are given negative values

Question 6

Constitutively Expressed Genes

Answer 6

• genes that are expressed through out the lifetime of a cell
• different genes can be expressed constitutively to different levels
• genes that are always expressed are referred to as vegetative or housekeeping genes, they usually encode proteins required for fundamental cell processes

Question 7

Consensus Sequence

Answer 7

• the best promoter sequence for binding
• 35Region ….16-19bp…..-10Region…..5-8bp….Inititation Site
• small changes in this sequence can effect the rate of transcription initiation

Question 8

Sigma Factor

Answer 8

• component of RNA polymerase
• determines protein specificity
• e.g. sigma 70 binds to -35 and -10 boxes of ‘vegetative’ or ‘housekeeping’ gene promoters
• after transcription initiated the sigma factor dissociates from the core RNA polymerase
• alternative sigma factors exist and they provide the cell with a mechanism for turning ON/OFF entire sets of genes depending on circumstances
• alternative sigma factors have different promoter specificities

Question 9

Ordered Expression of Sigma Factors Controls the Timing of Expression of T4 Lytic Genes - Sigma Factor Cascade

Answer 9

1) only a subset of the phages genes are initially transcribed by E.coli’s (host encoded) sigma 70
2) one of these early genes encodes an inhibitor (an anti-sigma factor) of sigma 70 which shuts off expression of host genes, another early gene encodes an alternative sigma factor
3) this alternatives sigma factor, sigma 33, directs transcription of a ‘middle’ set of genes primarily concerned with replication of the viral genome
4) ‘middle’ genes also include a gene encoding sigma 55
5) sigma 55 displaces sigma 33 (stopping genome replication) and directs transcription of the ‘late’ T4 genes which encode the structural proteins of the virus

Question 10

Transcriptional Termination

Answer 10

• in E.coli there are two classes of terminator sequence
  1) rho independent - consist of a G/C rich region of DNA whose base sequence is an inverted repeat to form a hairpin loop followed by a tun of Ts (Us in the RNA)
  2) rho dependent - form strong hairpin loops but not as strong as independent and without the UUUUUUU

Question 11

Bacterial Ribosomes

Answer 11

• ribosomes mediate translation
• bacterial ribosomes are 70S and comprise 2 major subunits, 30S and 50S
• the 30S subunit contains a 16S rRNA molecule
• the 50S subunit contains a 23S rRNA molecule

Question 12

Eubacterial Translation Initiation

Answer 12

• ribosomes start at the AUG (sometimes GUG/UUG) codon within 5 to 8 bp of a ribosome binding site on the mRNA (not just any AUG)
• ribosome binding site has a degree of complementarity to a segment of 16S rRNA, the RBS base pairs with the ribosome
• similarly to the consensus sequence can determine the efficiency of translation
• translation initiation is a major point of regulation in eubacteria
• segments encoding the RBS can be recognised in the DNA

Question 13

Translation

Answer 13

• base pairing sequence in mRNA or DNA sometimes called the Shine-Dalgarno sequence
• during translation the ribosome moves along mRNA in the 5’ to 3’ direction
• each triplet/codon of three bases codes for one amino acid
• polypeptide chain grows from its N terminus to its C terminus
• codon is recognised by an aminoacyl transfer RNA (tRNA) bearing the appropriate amino acid

Question 14

Translation Termination

Answer 14

• nascent polypeptide chain stays attached to the ribosome until translation terminates
• happens when the stop codon is reached
• ribosome, mRNA and polypeptide chain dissociate
• stop codons are recognised by release factors
• translational elongation and termination are not considered major points of regulation for gene expression

Question 15

Telomerase

Answer 15

• NA dependent, DNA polymerase

- reverse transcriptase

Question 16

Lac Operon - Genes

Answer 16

Lac Z - beta galactosidase, cytoplasmic enzyme that breaks down lactose to glucose and galactose
Lac Y - lactose permease, integral membrane protein that transports lactose across the cytoplasmic membrane
Lac A - transacetylase, may acetylate galactosidase sugars (other than lactose) preventing them becoming substrates for beta galactosidase

Question 17

Lac Operon

Answer 17

• single promoter and terminator
• promoter, operator, genes and terminator considered a part of the operon
• all 3 genes are cotranscribed to form a single mRNA, a polycistronic/polygenic mRNA
• this simple system ensures that all three genes are co-ordinately expressed

Question 18

Lac Operon - Controlling Elements and Genes

Answer 18

• gene expression is controlled at the level of transcription initiation by an adjacent gene lacI
• lacI encodes a repressor protein LacI
• lacI has its own promoter an terminator, they are not a part of the lac operon, they control it
• control genes are not always adjacent to the cognate operons although in the case of the lac operon lacI is adjacent

Question 19

Lac Operon - Order of Genes

Answer 19

-lacI is downstream of the operon
promoter-coding sequence-terminator
-after the lacI gene is the operon
promoter-operator-lacZ-lacy-lacA-terminator

Question 20

Induction

Definitoin

Answer 20

-production to a higher level

Question 21

Operon

Definition

Answer 21

• originally defined as a cluster of genes transcribed to produce a single mRNA molecule controlled by am operator, the binding site for a repressor
• later found that a promoter/operator mechanism may also control a single gene
• promoter may not be controlled by only one operator or even by any operator
• hence the term muti-gene-transcriptional unit - clusters of genes transcribed from a shared promoter

Question 22

Lac Operon - Negative Control

Glucose Present, Lactose Absent

Answer 22

• lacI is constitutively transcribed to produce a repressor protein
• the repressor attaches to the operator of the lac operon
• RNA polymerase is able to bind to the operon but cant initiate transcription
• the genes of the lac operon are not transcribed

Question 23

Lac Operon - Negative Control

Glucose Absent, Lactose Present

Answer 23

• lacI is constitutively expressed to produce a repressor protein
• inducer molecules, allolactose bind to the repressor causing a conformational change
• the inactivated repressor cannot bind to the operator of the lac operon
• RNA polymerase binds and transcribes the genes of the lac operon

Question 24

What does the lac operon explain?

Answer 24

• when E.coli is grown on a medium containing glucose and lactose it use up the glucose first
• only when the glucose has been depleted and growth is slowing do the cells start to use the lactose, and growth resumes
• this growth pattern is described as bi-phasic
• activity of beta galactosidase is ~1000x higher when the cells are using lactose rather than glucose
• in the absence of lactose there are only 1-5 molecules of the lac proteins per cell, after induction, up to 5000 molecules of beta galactosidase can accumulate within minutes

Question 25

Lac Operon | Why does the repressor not bind in the presence of lactose?

Answer 25

- residual levels of beta galactosidase (1-5 molecules per cell) exist in each cell - beta galactosidase catalyses a secondary reaction, the conversion of lactose to allolactose - allolactose can bind to the lac repressor - binding affects the conformation of LacI - the repressor loses its affinity for the operator/promoter of the lacoperon and releases from it - RNA polymerase is free to bind and initiate transcription of the lac operon - following induction, up to 5000 molecules of beta galactosidase can accumulate within minutes - the presence of lactose leads to the inactivation of the lac repressor and the induction of the lac operon

Question 26

Lac Operon - Positive Control

Answer 26

- occurs when glucose is absent, only results in higher transcription levels when lactose is present and not bound to the operator - when cAMP levels are high, cAMP binds to CAP (catabolite activator protein), this CRP (catabolite repressor protein) complex is then able to bind at a site upstream of the lac promoter - CRP can then contact RNA polymerase this contact increases the affinity of RNA polymerase for the lac promoter - in the absence of CRP at the upstream site, transcription from the lac promoter is lower as the lac promoter has poor -35 and -10 boxes - when glucose and lactose are both present, E.coli preferentially uses glucose due to low levels of cAMP - adding cAMP to cells restores the lac operon even when glucose is still present

Question 27

Catabolite Repression

Answer 27

- not only blocks activation of the lac operon | - blocks the uptake of lactose by LacY, i.e. it blocks induction, this is referred to as inducer exclusion

Question 28

General Conclusions From the Study of the Lac Operon

Answer 28

- sequence specific DNA binding proteins are key regulators of transcription - protein to protein interactions can either repress (repressor-RNA polymerase) or activate (CRP-RNA polymerase) transcription - small molecules can have key roles in gene expression by interacting with regulatory proteins - genetic and biochemical studies are needed to identify and characterise the interacting molecules - allolactose, DNA and tryptophan are all examples of small molecules that cause an allosteric change in DNA binding proteins - multiple signals integrated at the level of a single promoter - gene regularion and physiology are closely intertwined

Question 29

CRP

Answer 29

- capable of activating expression of >200 genes - including those for the utilisation of arabinose - shows same pattern as lactose - a global regulator, can bind to multiple promoters - no regulatory system is identical to the lac operon - great variety in modus operandi of transcription factors and their binding sits in DNA

Question 30

L-arabinose Catabolism

Answer 30

- shows induction and catabolite repression | - but in this case binding of inducers leads to the conversion of the repressor to an activator

Question 31

Regulon

Answer 31

- the entirety of all genes regulated positively (enhanced transcription) and/or negatively (reduced transcription) by a particular regulatory protein - expression of a particular regulatory protein allows co-ordinated regulation of multiple genes/operons (at differnet loci) in response to a common signal - e.g. SOS response

Question 32

SOS Response

Answer 32

- activated by DNA damage and coordinates various functions to allow and mediate repair - the SOS response is controlled by LexA - LexA represses a number of SOS genes through out the chromosome - various types of DNA damage cause regions of single stranded DNA - RecA binds to single stranded DNA regions, some repair is mediated by general recombination - this changes the conformation of RecA - RecA is then able to bind LexA, LexA then undergoes self proteolysis, cleaves itself and becomes inactive - LexA is no longer able to function as a repressor so the SOS genes can be expressed

Question 33

Gene Expression in E.coli and Other Bacteria

Answer 33

- bacteria respond to changes in the environment by altering the pattern of gene expression - the most common is control at the transcription initiation step - no other system is identical to the lac operon, there is a great variety in both cis and trans acting components - gene expression is also regulated by mRNA stability, translation, protein stability and post-translational modification - some genes are continuously expressed - all genes are regulated at some level

Question 34

Lac Operon - After Induction

Answer 34

1) lactose is cleaved and used as a carbon source 2) when cellular concentration of lactose falls, so does the concentration of allolactose 3) as cellular level of allolactose decreases, so do the number of repressors with allolactose bound to them 4) beta galactosidase can now split the release allolactose molecules for energy as well 5) repressor proteins (lacking allolactose) returns to the lac operon and rebinds 6) attachment of the repressor to the lac operon turns off transcription of the lac operon 7) production of the three gene products required for E.coli to utilise lactose are no longer synthesised 8) manufactured proteins remain in the cell until they are depleted as a consequence of cell division or degradation 9) some beta-galactosidase always exists as operators aren't occupied by the repressor proteins 100% of the time 10) beta galactosidase are instrumental in initial isomerisation of lactose to allolactose

Question 35

Lac Operon - Under Negative Control

Answer 35

-LacI blocks RNA polymerase if the inducer is absent

Question 36

Lac Operon | Repressor-Inducer Binding is in Equilibrium

Answer 36

- lots of lactose -> all repressor molecules bound by allolactose - moderat levels of lactose -> some repressor molecules bound by allolactose - very little lactose -> no repressor molecules bound by allolactose

Question 37

Lac Operon | How do we know that the lac operon isn't only negatively controlled?

Answer 37

-negative control does not explain why lactose isn't used when both glucose and lactose are present

Question 38

Lac Operon | How do we know that the lac operon is also positively controlled?

Answer 38

- not initially predicted - lac operon is only inducible in the absence of glucose - when E.coli is grown on glucose and lactose it uses glucose first, and only when the glucose has been depleted is the lac operon induced - catabolite (or glucose) repression, the lack of control at the level of transcription

Question 39

Lac Operon | Catabolite Repression and cAMP Production

Answer 39

- cAMP levels are related to the rate of glucose uptake - glucose uptake occurs by a phosphotransferase system which influences the activity of a membrane associated adenylate cyclase - in the absence of glucose uptake, adenylate cyclase is phosphorylated stimulating the conversion of ATP to cAMP

Question 40

Lac Operon | Activation of Transcription Initiation at the Lac Promoter by CRP-cAMP

Answer 40

- cAMP binds to CRP forming a cAMP-CRP complex - CRP can only bind to cognate sites when bound to cAMP - transcription initiation by RNA holoemzyme increases when it interacts on DNA with CRP

Question 41

Lac Operon - Promoter

Answer 41

- weak compared to consensus sequence - can be blocked easily - benefits from the assistance of an activation mechanism