Prokaryote Gene Structure, Regulation, and Information Transfer Flashcards

Question

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

Answer 1

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

Answer 2

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

Answer 3

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

Answer 4

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

Answer 5

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 2. 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) 3. 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.

Answer 6

- 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.

Answer 7

- 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).

Answer 8

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

Answer 9

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

Answer 10

Bacterial genome + Mobile Genetic Elements

Answer 11

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

Answer 12

Collection of Supergenomes within wider environment

Answer 13

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)

Answer 14

- 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

Answer 15

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

Answer 16

- 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)

Answer 17

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

Answer 18

- 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

Answer 19

o Larger chromosome = larger replication time = bad o Plasmids encoding different functions ‘distributed’ among different members of population.

Answer 20

- 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

Answer 21

- Determined by ori site 1. Theta replication 2. Rolling-circle replication

Answer 22

The process whereby plasmids transfer from 1 cell to another

Answer 23

Recipient cell + plasmid

Answer 24

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

Answer 25

- Made of protein called Pilin - Self-assembles - Forms cell membrane fusion - Functions in HGT

Answer 26

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

Answer 27

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

Answer 28

o Recombination between shared sequences on plasmid and on chromosome o Recombination via insertion elements shared between chromosome and plasmid

Answer 29

Plasmid integrated into a chromosome

Answer 30

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.

Answer 31

- 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

Answer 32

Cell formed from F' x F- conjugation Has multiple copies of the same gene

Answer 33

- 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.

Answer 34

- 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

Answer 35

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

Answer 36

- 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)

Answer 37

Bacteriophage cell that includes fragments of degraded bacterial genome

Answer 38

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

Answer 39

- 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.

Answer 40

Movement of transposon from one location to another

Answer 41

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

Answer 42

- 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

Answer 43

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

Answer 44

- 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

Answer 45

- 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'

Answer 46

- 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

Answer 47

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

Answer 48

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

Answer 49

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

Answer 50

A gene that is expressed continuously

Answer 51

Genes that are specifically activated for expression (default state is 'off') Activated by presence of an inducer that allows for gene expression

Answer 52

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

Answer 53

Increased synthesis of genes in response to an inducer

Answer 54

Decreased synthesis of genes in response to an inhibitor/corepressor

Answer 55

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

Answer 56

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

Answer 57

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

Answer 58

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

Answer 59

- 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

Answer 60

- 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.

Answer 61

- 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

Answer 62

- 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

Answer 63

- 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.

Answer 64

- 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 )

Answer 65

- 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

Answer 66

- 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

Answer 67

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

Answer 68

- 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

Answer 69

An Activator regulatory protein for the lac operon

Prokaryote Gene Structure, Regulation, and Information Transfer Flashcards

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