Bacterial Gene Expression 1 Flashcards
Fundamental Issues of Gene Transcription
- 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
Constitutive Gene Expression
-genes expressed throughout the life time of the cell
Methods of Specific Protein Levels Control
- 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
Transcription
- 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
Labelling Positions in the DNA Sequence
- 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
Constitutively Expressed Genes
- 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
Consensus Sequence
- 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
Sigma Factor
- 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
Ordered Expression of Sigma Factors Controls the Timing of Expression of T4 Lytic Genes - Sigma Factor Cascade
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
Transcriptional Termination
- 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
Bacterial Ribosomes
- 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
Eubacterial Translation Initiation
- 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
Translation
- 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
Translation Termination
- 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
Telomerase
- NA dependent, DNA polymerase
- reverse transcriptase
Lac Operon - Genes
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
Lac Operon
- 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
Lac Operon - Controlling Elements and Genes
- 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
Lac Operon - Order of Genes
-lacI is downstream of the operon
promoter-coding sequence-terminator
-after the lacI gene is the operon
promoter-operator-lacZ-lacy-lacA-terminator
Induction
Definitoin
-production to a higher level
Operon
Definition
- 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
Lac Operon - Negative Control
Glucose Present, Lactose Absent
- 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
Lac Operon - Negative Control
Glucose Absent, Lactose Present
- 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
What does the lac operon explain?
- 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