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MCB2020F > Prokaryote Gene Structure, Regulation, and Information Transfer > Flashcards

Prokaryote Gene Structure, Regulation, and Information Transfer Flashcards

1
Q

What is a gene?

A
  • The basic unit of genetic information

- A polynucleotide sequence that codes for a functional product

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2
Q

What is a reading frame?

A
  • Gene sequence that gives rise to a single protein product
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3
Q

What is a codon?

A

Discrete 3-nucleotide sequence on an mRNA molecule that codes for a single amino acid

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4
Q

Bacterial DNA structure

A
  • DNA is double stranded and directional
  • Coding strand (sense strand) = 5’ – 3’strand
  • Template strand (antisense strand) = 3’ – 5’ strand
  • DNA Has promoter and leader regions on left of coding region
  • DNA Has trailer and terminator regions on right of coding region
  • RNA Polymerase recognizes template strand.. DOESN’T bind to coding strand
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5
Q

Bacterial Promoter Region Structure

A

o Located at start of gene

o Operator site is a sequence recognized by DNA binding proteins specific for that sequence; has no fixed position relative to promoter

o ‘+1’ in promoter region signals transcriptional start

o 5’ end of ‘+1’ is called upstream

o ‘-35’ (no. of nucleotides away from +1) in promoter region is RNA polymerase recognition site

o ‘-10’ (no. of nucleotides away from +1) in promoter region is RNA polymerase binding site. Also called the PRIBNO BOX

o -10 & -35 sequences are called consensus sequences

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6
Q

Bacterial mRNA structure

A
  • Leader region:
    o Shine-Dalgarno sequence is a purine rich region to which ribosome binds (in mRNA)
  • Promoter, leader, trailer and terminator regions aren’t translated (UTR untranslated regions)
    o Leader and trailer are transcribed but NOT translated
  • has Leader and trailer regions, but NOT promoter and terminator regions.
  • AUG is start codon for translation
  • 3 translation stop Codons: UAA, UGA, UAG
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7
Q

What is a bacterial operon?

A

A cluster of genes controlled by the same regulatory control (same promoter region)

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8
Q

What is polycistronic mRNA?

A
  • Basically 1 strand of mRNA with many genes on it
  • Very common in bacteria
  • Separate genes on operon are transcribed as part of a single mRNA transcript (from 1 long mRNA strand)
  • Different reading frames translated into different proteins
  • Intercistronic spacer regions separate the individual coding segments
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9
Q

In what direction does RNA polymerase read the template DNA strand during transcription?

A

3’-5’ direction

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10
Q

What types of RNA are produced by transcription?

A
  1. mRNA carries transcript for protein synthesis
  2. tRNA carries amino acids during protein synthesis
  3. rRNA mols are components of ribosomes
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11
Q

What are the 2 types of bacterial mRNA transcripts?

A
  • Monocistronic/monogenic (single gene transcribed)

- Polycistronic/polygenic (multiple genes transcribed)

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12
Q

RNA Polymerase enzyme structure

A
  • 2 alpha, 2 beta, 2 omega subunits = core enzyme (cannot bind DNA tightly or specifically)
  • Holoenzmye = when core enzyme binds to sigma factor
  • Sigma factor: recognizes and binds to promoter region
  • Alpha factor: chain initiation and interaction with regulatory proteins
  • Beta Factor: chain initiation and elongation
  • Beta+ factor: DNA binding
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13
Q

Prokaryote transcription initiation

A
  • Binding of the sigma factor of RNA Polymerase
  • Promoter is recognition/binding site for RNA polymerase. It functions to orient polymerase for initiation
  • RNA Pol only binds at -10 site if it is associated with a -35 sequence with the right distance between them
  • RNA Pol cannot bind directly to sequence, has to bind via sigma factor
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14
Q

What is the effect of promoters on the efficiency of transcription?

A
  • Strong promoters tend to have unaltered consensus sequences
  • Weak promoters have substitutions within this region
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15
Q

Prokaryote Transcription Elongation

A
  • RNA Pol holoenzyme unwinds DNA to form transcription bubble (Open Promoter Complex)
  • RNA Pol Transcription Bubble moves in 3’-5’ direction causing sigma factor to dissociate from core enzyme
  • No primer required, within bubble a temp RNA:DNA hybrid is formed
  • Temp DNA strand is used to make complimentary mRNA
  • Ribonucleotides added via phosphodiester bonds at 3’ end of RNA
  • Transcription bubble moves @ 50 nucleotides/s
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16
Q

Prokaryote Transcription Termination

A
  • Hairpin loop followed by A-U rich sequence in the trailer and terminator region marks termination
  • 2 methods of termination
  1. Rho-independent termination (intrinsic terminators):
    • Hairpin loop structure stalls RNA polymerase
    • Weak A-U bonds are not able to hold RNA:DNA hybrid and RNA Pol falls off (mRNA forced out of Pol complex)
  2. Rho-dependant termination (extrinsic terminators):
    • Requires the aid of the rho factor
    • RNA Pol stalls at hairpin loop
    • rho factor binds to rut site (a cis sequence, i.e. doesn’t produce protein) on the mRNA and moves to stalled RNA Pol and separates it from DNA:RNA hybrid
  • Often multiple hairpin loops are needed to stop termination
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17
Q

What is Rho-independent Termination?

A
  • Method of Prokaryotic termination
  • Hairpin loop structure stalls RNA polymerase
  • Weak A-U bonds are not able to hold RNA:DNA hybrid and RNA Pol falls off (mRNA forced out of Pol complex)
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18
Q

What is Rho-dependant Termination?

A
  • Requires the aid of the rho factor
  • RNA Pol stalls at hairpin loop
  • rho factor binds to rut site (a cis sequence, i.e. doesn’t produce protein) on the mRNA and moves to stalled RNA Pol and separates it from DNA:RNA hybrid
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19
Q

What is code degeneracy?

A
  • Up to 6 different codons can code for a single amino acid
  • Referred to as ‘Wobble’
  • Result of Loose base pairing
    • Because the 3rd base less important than first 2 bases
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20
Q

How many sense codons are there?

A

61 codons that specific amino acids

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21
Q

How many Stop codons are there?

A

3, UAA, UAG, UGA

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22
Q

What is the importance of Open Reading Frames (ORFs)?

A

Even a single change in a reading frame can create a totally new message

i.e. code a totally new product

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23
Q

What is protein synthesis?

What is its direction of synthesis?

What is a polyribosome?

A
  • The synthesis of polypeptides directed by a sequence of nucleotides in mRNA
  • N terminal -> C terminal
  • Complex of mRNA with several ribosomes
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24
Q

Structure of the prokaryote ribosome

A
  • 70S ribosome
  • 30S subunit = 16S rRNA + 21 polypeptide chains
  • 50S subunit = 5S rRNA + 23S rRNA (peptide transferase) + 34 polypeptide chains
  • 3 distinct binding sites (A,P,E)
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25
Q

What are the 3 binding sites of the 70S ribosome?

A
  1. A (Acceptor) site: attachment site for incoming aminoacyl-tRNA
  2. P (Peptidyl) site: Occupied by tRNA carrying growing peptide chain
  3. E (Exit) site: Transiently occupied by deacetylated tRNA
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26
Q

What are aminoacyl-tRNA’s?

A

Complex of tRNA bonded to an amino acid via an ester bond on the 3’ end of tRNA

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27
Q

What is aminoacylation?

A

The ATP-dependant attachment of a specific amino acid onto the 3’ end of a tRNA, catalysed by Aminoacyl-tRNA synthetases.

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28
Q

Prokaryotic Translation Initiation

A

Summary: Binding of mRNA and initiator aminoacyl-tRNA to ribosomal small subunit, followed by binding of large subunit

  • Coding region of open reading frame:
    o Start codon 5’-AUG-3’
    codes for N-formylmethionine (f-Met-tRNA(i)(Met)) – a modified amino acid used for initiating protein synthesis.
    o Met-tRNA(m)(Met) used internally to code form Methionine
    o N-terminal Met often removed post-translationally
  • 30S ribosome complex formation:
    o 30S initiation complex = Initiation factor proteins (IF’s), GTP, N-formyl-Met-tRNA(i)(fMet), mRNA, 16S RNA of 30S subunit
    o Shine-Dalgarno sequence complementary to 16S RNA sequence – allows proper alignment of ribosome on mRNA with respect to start codon.
    o f-Met-tRNAifMet gets bound at P site via IF’s to the AUG codon
  • 70S ribosome formation:
    o Initiation factors released, leads to 50S binding to 30S complex
    o The A site now poised to accept incoming aminoacyl-tRNA
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29
Q

Prokaryotic Translation Elongation

A

Summary: Movement of ribosome along mRNA and synthesis of all peptide bonds – with tRNAs bound to acceptor (A) and peptidyl (P) sites

  • 3 principle simultaneous steps:
    1. Codon directed binding of incoming aminoacyl-tRNA at A-site
  1. Peptide bond formation: transfer of peptidyl chain from tRNA in P-site to N-terminus of tRNA in A-site
    i. Done by 23S rRNA (Peptidyl transferase)
  2. Old tRNA moves to E-site and is released when a new aminoacyl-tRNA binds to A-site. New tRNA (now carrying the peptide chain) moves into P-site.
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30
Q

Prokaryotic Translation Termination

A
  • Coding region on mRNA ends with stop codon followed by trailer sequence
  • Stop codons not read by any tRNAs
  • Release factors (proteins) recognize stop codon at A site
  • Presence of release factors with a stop codon at A-site allows cleavage of tRNA carrier from peptide chain.
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31
Q

What is Translational Coupling in Polycistronic mRNA?

A
  • Polycistronic mRNA has multiple operons, each with their own leader regions and Shine-Dalgarno’s
    o This means that theoretically multiple ribosomes could bind to the mRNA and form multiple proteins simultaneously
  • However in translational coupling each gene requires the preceding gene to be translated first
  • Initiation codon and Shine-Dalgarno regions get ‘hidden’ in a hairpin loop, so the Ribosome cannot bind to it – translation cannot happen
    o Ribosome can only bind to linear structure
  • As the ribosome moves along mRNA towards next gene, hairpin loop unfolds so that AUG codon is available for translation to occur (By a different ribosome).
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32
Q

What are the advantages of translational coupling of polycistronic mRNA?

A

Allows for:

  • Spatial and temporal expression of genes (proteins are only made when they are required at a particular time and for a particular process)
  • Different amounts of proteins to be translated from the same polycistronic operon
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33
Q

What is Horizontal Gene Transfer?

A

Transfer of genes between the total genome, super genome, and meta genome by Mobile Genetic Elements

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34
Q

What is the Total Genome?

A

Bacterial genome + Mobile Genetic Elements

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35
Q

What is the Supergenome?

A

Total number of genes available to a bacterial community via MGEs within the same environment

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36
Q

What is the Metagenome?

A

Collection of Supergenomes within wider environment

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37
Q

What is a plasmid?

A

DNA molecule that exists separately from chromosome and contains an origin of replication – it can replicate independently

  -	However, it does need the machinery of the cell. And replicates when bacteria replicates (not whenever it wants to)
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38
Q

What are the features of plasmids?

A
  • Vary in size (<2000 bp to > 100 000 bp)
  • Exist either as circular dsDNA (common) or linear dsDNA (rare)
  • Have specific copy number: Low copy (1-15) vs High copy (>50)
  • Faithfully transmitted to daughter bacterial cells
  • Encode non-essential but beneficial products
39
Q

What are the 6 types of plasmid?

A
  1. Fertility (F) plasmids can direct conjugation
  2. Resistance (R) plasmids: genes conferring resistance to antibacterial agents.
  3. Col plasmids: genes for colicins (Produce proteins that kill other bacteria, often of same species)
  4. Metabolic (Degradative) plasmids: carry genes for metabolism of unusual molecules
  5. Virulence plasmids: confer pathogenicity on the host bacterium
  6. Cryptic: no obvious phenotype
40
Q

Features of the F Plasmid

A
  • Circular plasmid
  • oriV = site of replication
  • oriT = initiation site for conjugal transfer
  • sequences regulate insertion into bacterial genome (can jump in and out of bacterial genome)
41
Q

Current favourite hypothesis for the origin of plasmids

A

They were facultative symbionts that provided their prokaryotic hosts with means of obtaining increased fitness at a trade-off of the resources required for their own existence

42
Q

Plasmid Functions

A
  • Plasmid genes usually give bacteria a selective advantage under only some conditions
  • Tend to carry non-essential genes
  • Gene products:
    o Utilization of unusual carbon sources (toluene)
    o Resistance to heavy metals, antibiotics
    o Synthesis of toxins
43
Q

Why are Plasmid traits carried on plasmid not chromosome?

A

o Larger chromosome = larger replication time = bad

o Plasmids encoding different functions ‘distributed’ among different members of population.

44
Q

Plasmid Structure

A
  • Covalently closed circular:
    o Nucleotides in each strand joined covalently
    o Prevents strands from separating
    o Causes DNA to coil up (internal tension) = negative supercoiling
    o Remaining stress helps separation of strands for replication or transcription
  • Linear dsDNA:
    o Not well characterized
    o Covalently closed hairpins at ends to protect against exonucleases and for replication
45
Q

2 Methods of Plasmid Replication

A
  • Determined by ori site
  1. Theta replication
  2. Rolling-circle replication
46
Q

What is bacterial conjugation?

A

The process whereby plasmids transfer from 1 cell to another

47
Q

What is a transconjugant?

A

Recipient cell + plasmid

48
Q

3 classification of plasmid based on their transfer ability

A
  1. Non-transmissible plasmids:
    a. Lack genes for effective contact and DNA transfer
  2. Self-transmissible plasmids (conjugative & mobilizable) – e.g. F plasmids
    a. Encode all functions (full set of tra genes) needed to make contact & transfer DNA between cells
    b. Can also aid in transfer of mobilizable plasmids
  3. Mobilizable plasmids (non-conjugative & mobilizable)
    a. Cannot make contact
    b. Encode some but not all proteins required for transfer
    c. Can prepare its DNA:
    i. Has tra E genes but no genes for sex pilus (tra A&B) for transfer
    ii. Needs help of self-transmissible plasmids to transfer
49
Q

What is a sex pilus?

A
  • Made of protein called Pilin
  • Self-assembles
  • Forms cell membrane fusion
  • Functions in HGT
50
Q

Method of F Plasmid Conjugation

A
  1. Donor (F+, means has F plasmid) produces sex pilus to contact recipient (F-, doesn’t have F plasmid)
  2. Once contact is made, pilus retracts to bring F- closer (recognizes F- because of receptors on cell mem)
  3. Mating pair formation (conjugation bridge/pore) forms between F+ & F- cell membranes
    a. Allows DNA to pass through membranes, sex pilus only brings them closer
  4. 1 of DNA strands on F plasmid is nicked at oriT and separates
  5. Intact F plasmid is replicated in F+ cell, 5’ end of nicked DNA passes into F- cell via the pore.
  6. As the ssDNA of F+ enters F- its replicated to become dsDNA producing a F plasmid copy (i.e. F- becomes F+). F plasmid circularizes/ligates -> circular chromosome.
  7. F+ plasmids produce surface exclusion proteins which prevent the donors from conjugating with other F+ plasmids
    a. TraT blocks mating pair formation
    b. TraS blocks DNA transfer at the pore
51
Q

What is Hfr Conjugation?

A

Bacterial conjugation of Hfr strain (bacteria with integrated F plasmid) and an F- cell.

Has very high efficiency

52
Q

How can plasmid integration occur into a chromosome?

A

o Recombination between shared sequences on plasmid and on chromosome

o Recombination via insertion elements shared between chromosome and plasmid

53
Q

What is an episome?

A

Plasmid integrated into a chromosome

54
Q

Method of Hfr conjugation

A
  1. Sex pilus of Hfr cell (integrated F plasmid) establishes connection with F- cell
  2. Pore is formed, single strand of F plasmid is cut and starts to enter the F- cell
  3. Because it’s in the chromosome, the chromosomal strand enters the cell behind it
    a. Theoretically whole chromosome could cross over, but because the pore is very delicate in practice it’s impossible for the entire chromosome to transfer
  4. Hfr chromosome replicates itself as transfer proceeds
  5. Mating pair formation breaks, Cells separate – piece of F plasmid + part of bacterial chromosome in F- cell.
  6. Donor DNA and recipient DNA will have some of the same genes (homologous regions). Crossovers can occur
  7. Some of transferred DNA may be incorporated into recipient’s chromosome by recombination
  8. Cell remains F- but carries some genes from Hfr chromosome
  9. Nucleases degrade part of F plasmid that was transferred and left-over DNA fragments.
55
Q

What is F’ conjugation?

A
  • F episome can excise itself from the chromosome of Hfr cell to reform the F plasmid
  • An error can occur during excision and plasmid can pick up portion of the chromosome
  • Genotypically distinct from F plasmid – now called F’ plasmid
  • F’ X F- can transfer bacterial genes acquired during excision:
    o Forms partial diploid merozygote (will have multiple copies of same gene)
    o Same bacterial gene present on F’ plasmid also found on recipient chromosome
56
Q

What is a partial diploid merozygote?

A

Cell formed from F’ x F- conjugation

Has multiple copies of the same gene

57
Q

What is plasmid addiction?

A
  • Found on low copy number plasmids
  • One of many mechanisms to ensure the plasmid is not ‘lost’ from the population
    o Ensures the daughter cell will have a copy of the plasmid
  • Requires a stable (long half-life) toxin (can kill cell) produced in low amounts and an unstable (short half-life) antitoxin (can save cell) produced constantly in large amounts by the plasmid.
    o Toxin targets vital host protein, loss of its function will kill the cell, so needs antitoxin to be constantly produced.
    o Antitoxin continually degraded by host proteases and is made in excess, so toxin is inactivated before it can attack host target.
  • If cell is plasmid-cured (loses its plasmid) no new toxin & antitoxin is produced. Toxin will survive longer so it will attack the host target and the cell will die.
58
Q

What is transduction?

A
  • Transfer of bacterial genes by phages.
  • Bacterial genes incorporated into a phage capsid due to errors made during the virus lifecycle
  • Phage containing these genes then injects them into another bacterium
59
Q

Bacteriophage lytic cycle

A
  1. Absorption & penetration: phage attaches to specific receptor and injects dsDNA into cell
  2. Early mRNA is formed
  3. Host DNA degraded by enzymes
  4. Phage forces bacteria to make virus DNA & proteins
  5. Late RNA formed, Heads & tails made
  6. Phages assembled where virus DNA packed within virus protein coat
  7. Mature virus released by cell lysis
  8. Phages that reproduce using a lytic cycle = virulent phages
60
Q

Process of Generalised Transduction

A
  • Occurs during the lytic cycle
  • Phage DNA enters host, bacterial chromosome degraded
  • During packaging of viral chromosome into capsid, random fragments of degraded bacterial genome can also be packaged by mistake -> Generalized transducing particle (no longer a phage)
  • Random bacterial DNA injecting into another cell by transducing particle (No initiation of lytic cycle)
  • 3 routes for injected dsDNA:
    o Integration into host genome – stable
    o Degradation – recognized as foreign by restriction enzymes and cut out of DNA and degraded
    o Non-integration and expression = abortive transductions (partial diploids); temporary (lost during cell division)
61
Q

What is a generalised transducing particle?

A

Bacteriophage cell that includes fragments of degraded bacterial genome

62
Q

What is transformation?

A

The uptake by a ‘competent’ cell of a DNA mol or fragment from the medium into the cell

63
Q

Process of transformation

A
  • Bacterial cells have specific DNA uptake mechanisms, which allow the bacterial cells to take up DNA from the medium
  • The uptake mechanisms are turned on when a cell undergoes stress
  • A DNA fragment in the medium binds to a cell surface receptor associated with the DNA uptake system
  • An extracellular endonuclease cuts the DNA into smaller fragments
  • 1 strand is degraded, and a single strand is transported into the cell
  • The single strand aligns itself with the homologous region on the bacterial chromosome
  • The DNA strand is incorporated into the chromosome via homologous recombination
  • The heteroduplex DNA is repaired in such a manner that the original genotype of the chromosomal gene is changed to the genotype of the incoming strand.
64
Q

What is transposition?

A

Movement of transposon from one location to another

65
Q

What is a transposon?

A

Pieces of DNA that can move around the genome of an individual cell. Can also be transferred to other cells.

66
Q

Effects of transposition

A
  • Insertion into a gene can cause mutation or DNA rearrangement (deletion of genetic material)
  • Some transposons carry stop codons or termination sequences – block translation or transcription respectively
  • Some carry promoters that can activate genes near point of insertion -turn genes on or off
    o Contributes to genetic diversity by producing new phenotypes
  • Transposons on plasmids play a role in plasmid function and evolution: transferring antibiotic resistance
67
Q

Types of Class 1 Transposons

A
  1. Insertion sequences
  2. Composite Transposons
  3. Replicative (non-composite) Transposons
  4. Conjugative transposons
68
Q

Insertion Sequences

A
  • Carries only genes for movement – transposase
    o Flanking direct repeats and inverted repeats on either side of gene
  • Each type designated by prefix ‘IS’ followed by a number
  • Commonly moves via Nonreplicative transposition (cut-and-paste)
    o Transposase enzyme recognizes inverted repeats and excises the transposon
  • Generates flanking direct repeats at site of insertion
  • No increase in copy number of IS
69
Q

Composite Transposons

A
  • DNA segment that is flanked by two copies of an Insertion Sequence (IS)
  • DNA segment not required for movement and carries additional information (e.g. antibiotic resistance)
  • Transposase of one IS sequence catalyzes transposition of both IS elements including DNA between them (cut-paste)
  • Also generates flanking direct repeats at site of insertion

‘DFR - IR - IS - IR - DNA segment - IR - IS - IR - DFR’

70
Q

Replicative (Non-composite) Transposons

A
  • Has inverted repeats but not IS elements at their ends
  • Encodes a transposase and resolvase and has a res site
  • Carry genes for antibiotic resistance (AbR), virulence factors and catabolic enzymes
  • Replicative transposition (copy-and-paste): original transposon remains at parental site on chromosome while replicated copy inserts at target DNA
  • Copy number of replicative transposons increases after transposition

‘DFR - IR - Transposase - Res site - Resolvase - Abr - IR - DFR

71
Q

Conjugative Transposons

A

A. Plasmid independent:

a. Nothing is known
b. Has tra genes and can form circular intermediate

B. Plasmid dependent:

a. Reside on conjugative or mobilizable plasmids and are transferred via conjugation with a conjugative plasmid
b. Many transposons are found within each other on a plasmid
c. Responsible for conferring antibiotic resistance
72
Q

Levels of Bacterial Gene Regulation

A

Transcription:
- Genetic regulatory proteins can bind to DNA and control whether or not transcription begins [Same in Archaea]
- Attenuation: transcription can terminate very early due to formation of a transcriptional terminator
o Binding of a metabolite to a riboswitch in mRNA can cause premature termination

Translation:

  • Translational repressor proteins can bind to mRNA and prevent translation from starting
  • Antisense RNA can bind to mRNA and control whether translation begins
  • Binding of metabolite to a riboswitch can block translation [Same in Archaea]

Post translation: [Same in Archaea]
- Small mols can bond non-covalently to a protein and affect its function
- Structure and function of a protein can be altered by covalent changes in protein
o Can be reversible or irreversible

73
Q

Levels of Gene Regulation in Eukarya

A

Transcription:

  • Regulatory transcription factors may activate or inhibit transcription
  • Compaction level of chromatin influences transcription
  • DNA methylation usually inhibits transcription

RNA processing:

  • Alternative splicing alters exon choices
  • RNA editing alters the base sequence of mRNAs

Translation:

  • Translation may be regulated by phosphorylation of translational initiation factors
  • Translation may be regulated by proteins that bind to the 5’ end of the mRNA
  • Antisense RNA can bind to mRNA and control whether or not translation begins
  • mRNA stability may be influenced by RNA binding proteins.

Post translation:
- Feedback inhibition and covalent modifications may regulate protein function

74
Q

What is a constitutive gene?

A

A gene that is expressed continuously

75
Q

What are inducible genes?

A

Genes that are specifically activated for expression (default state is ‘off’)

Activated by presence of an inducer that allows for gene expression

76
Q

What are repressible genes?

A

o Default state is on

o Gene expression is specifically repressed by the presence of a inhibitor/corepressor that suppresses gene expression

77
Q

What is induction?

A

Increased synthesis of genes in response to an inducer

78
Q

What is repression?

A

Decreased synthesis of genes in response to an inhibitor/corepressor

79
Q

What are regulatory proteins?

A

o Activators promote/induce transcription = positive control

o Repressors repress/inhibit transcription = negative control

o They can bind DNA

o They are regulated allosterically: activity is modulated by the binding of inducers and corepressors/inhibitors

80
Q

Inducers, Inhibitors & co-repressors

A

o Can be small effector proteins or metabolites

o Non-covalently bind to regulatory proteins

o Change the activity of the regulatory protein (active or inactive)

o Interactions between effector and regulatory proteins affect transcription

81
Q

What is a cis-regulatory element?

A

A region of DNA or RNA that is involved in regulating the expression of genes located on the same molecule

e.g. DNA: Operator, activator, consensus sequences
RNA: Shine-Delgarno, RIboswitch

82
Q

What are trans-regulatory elements?

A

Product of a gene which may modify (or regulate) the expression of distant genes(s)

83
Q

Positive Control of an inducible gene

A
  • Absence of an inducer means activator protein cannot bind to activator binding site, RNA Pol cannot bind to promoter, transcription doesn’t happen
  • Inducer binds to activator protein – allosterically changes shape of activator and allows it to bind to activator binding site on gene
  • Allows RNA Pol to bind to promoter of mRNA and transcription occurs
84
Q

Positive control of a repressible gene

A
  • Inhibiter binds to activator protein – changes allosteric conformation of activator protein causing release from activator binding site
  • RNA Pol can no longer bind to the promoter, transcription stops.
85
Q

Negative control of an inducer gene

A
  • Repressor protein bonded to operator site, blocks RNA Pol by blocking promoter
  • Inducer changes allosteric conformation of repressor and it releases from operator site – RNA Pol gains access to promoter for transcription
86
Q

Negative control of a repressible gene

A
  • Repressor unable to bind to operator site – transcription occurs
  • Inhibitor (corepressor) causes allosteric conformational change in the repressor protein so it binds to the operator site and blocks RNA Pol from the promoter – blocks transcription
87
Q

Methods of regulation at Bacterial Transcriptional Initiation

A
  • Nucleotide sequence of promoter (cis-regulatory element) is important:
    o Determines the strength and stability of binding by RNA polymerase to the promoter region
    o A poor consensus sequence will result in weak binding by RNA polymerase and in weak transcription = very few copies of mRNA transcript produced.
  • Bacterial sigma factors:
    o Required by RNA polymerase for transcriptional initiation
    o Different types of sigma factors required for transcription under specific environmental or physiological conditions
    o Allows genes and operons to be controlled globally under certain conditions
  • Repressor & activator proteins:
    o Their presence or absence affects transcription by RNA polymerase
    o These trans-regulatory elements binding to the promoter region could enhance or prevent RNA polymerase binding.
88
Q

Post-Transcriptional Regulation

A
  • mRNA stability:
    o Structure of mRNA can result in mRNA half-life being long or very short
    o Results in short or long translation window for the ribosome
    (Short mRNA half-life = very few proteins produced )
89
Q

Methods of Translational Regulation

A
  • Nucleotide sequence of Shine-Delgarno is important:
    o Determines the strength of binding by Ribosome complex
    o A poor Shine Delgarno sequence will result in weak binding of the ribosome = very little protein produced
  • Small RNAs (noncoding RNAs)
    o Range in size from 40-600 nucleotides
    o Antisense RNA
    o Some sRNAs base pair to the leader region of target mRNAs blocking ribosome binding and therefore translation
    o Some sRNAs promote translation upon binding to the mRNA by changing its structure
    o Most sRNAs require chaperone proteins
90
Q

Translational Regulation by Riboswitch

A
  • Translation occurs to make a protein which results in the synthesis of a specific product (ligand) which controls the proteins expression
    o i.e. it regulates its own activity
  • Upstream of the Shine Delgarno is the riboswitch region in the form of a hairpin loop that has a binding site for the ligand
  • The loop has a region which is complementary to the Shine Delgarno but is sequestered by complimentary base pairing in the loop
  • If sufficient ligand is made then it binds to the hairpin loop and disrupts its formation resulting in the complimentary Shine Delgarno binding to the Riboswitch and preventing the ribosome from binding – no translation occurs
91
Q

What is catabolite repression?

A

When the synthesis of enzymes involved in the breakdown of other sugars is prevented by the preferred sugar

92
Q

What is diauxic growth?

A
  • A biphasic growth pattern in which there is a preferential use of one carbon source over another when both are available in the environment
  • Lag occurs after preferred substrate is exhausted followed by the resumption of growth using the second source
93
Q

What is the Catabolite Activator Protein (CAP)?

A

An Activator regulatory protein for the lac operon

MCB2020F (6 decks)

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