Microbiology Module 4 Quiz

Order of elements in a bacterial gene (5′→3′)

Promoter → +1 → leader (SD site) → start codon → coding sequence → stop codon → trailer → terminator

Promoter: function

DNA sequence that tells RNA polymerase where to start transcription and how often (closer to consensus = more transcription).

Promoter: location and what binds

upstream of +1 (-35 and -10 boxes); never transcribed; binds sigma factor, as part of RNA polymerase

What is an operator

Genes with related functions under one promoter, made as one polycistronic mRNA

Operator: function

regulatory DNA site that lets the cell switch an operon on or off, when a repressor sits on it, RNA polymerase is blocked

Operator: location and what binds

near or overlapping the promoter, binds repressor proteins (e.g., LacI, TrpR)

+1 site: function

marks the exact base where transcription begins; it is the first nucleotide copied into RNA (the 5′ end of the mRNA)

+1 site: location and what binds

just downstream of the promoter, binds RNA polymerase which starts synthesis

Shine-Dalgarno (SD) site: function

ribosome-binding site that lines the ribosome up so the start codon lands in the P site

Shine-Dalgarno (SD) site: location and what binds

in the leader of the mRNA (transcribed, not translated), a few bases upstream of AUG; binds 16S rRNA of the 30S subunit

Start codon: function

signals where translation begins and sets the reading frame; codes for the first amino acid (fMet)

Start codon: what binds

anticodon of fMet-tRNA in the P site

Protein coding sequence: function

the codons that specify the amino acid sequence of the protein

Protein coding sequence:

between start and stop codons; read by ribosomes, tRNA anticodons pair with each codon

Stop codon: function and binds with

signals the end of translation, but no tRNA reads it/no amino acid is attached to it

Stop codon: sequences

UAA, UAG, or UGA

Terminator: function

signals RNA polymerase to stop transcribing and release the RNA

Terminator: location

downstream of the trailer

2 methods that signal RNA polymerase to terminate

Rho-independent or Rho-dependent

Rho-independent

RNA polymerase slows down in hairpin + run into U’s (which are weakly bound) and pops off

Rho-dependent

Rho binds rut site on RNA, chases the paused polymerase, and uses helicase activity to pull the RNA off

Which regions are transcribed but not translated?

Leader and trailer

tRNA

the adapter that matches a codon to its amino acid

What are the 2 parts of a tRNA

Adapter and anticodon

What is the adapter part of tRNA

acceptor (3′ CCA) end carries the amino acid

What is the anticodon part of tRNA

pairs with the codon

rRNA

Ribosomes are built from rRNA plus proteins, and the rRNA makes up the core of the machine

it gives the ribosome its structure, helps it bind mRNA and tRNA, and carries out the key chemical step of linking amino acids together

16S rRNA

In the 30S subunit. Binds the SD sequence (initiation) and the 3′ CCA end of aminoacyl-tRNA

23S rRNA

In the 50S subunit. A ribozyme that catalyzes peptide bond formation

What is 70S

The whole bacterial ribosome (30S + 50S), not an rRNA

Sigma factor vs. core enzyme

the first finds the promoter, the later builds RNA

together = holoenzyme

What happens to sigma after initiation?

Leaves after ~12 nucleotides, because its job (promoter recognition) is done

Consensus sequence and promoter strength

Ideal promoter sequence. Closer to consensus = stronger sigma binding = more transcription

Vertical gene transfer

parent to offspring

Horizontal gene transfer

between independent organisms, independent of reproduction

Four outcomes of transferred DNA

(1) Integrates → stable. (2) Self-replicates (plasmid) → stable. (3) Can’t replicate → lost. (4) Degraded by restriction enzymes/CRISPR → lost

Conjugation

direct transfer of DNA from a donor cell to a recipient cell through physical contact; Requires cell-cell contact and a conjugative plasmid (F factor)

Transformation

Uptake of free DNA from the environment. Some bacteria are naturally competent

Generalized transduction

A phage accidentally packages bacterial DNA; any region can transfer

Specialized transduction

Lysogenic phage excises imprecisely; transfers genes next to the integration site

Genomic island: definition and how identified

Region with abnormal %GC vs. the rest of the genome; likely acquired by HGT (horizontal gene transfer)

Possible roles of genomic islands

pathogenicity island, genomic island, symbiosis island, resistance island, metabolic island

Role of secretion systems

Move proteins from the cytoplasm across membranes (or into target cells)

Which systems work in Gram+ / Gram+ and Gram−

Sec and Tat

Which systems work in Gram−

Types I-VI

Sec

transfers unfolded proteins

Tat

transfers fully folded proteins

YidC

folds and inserts membrane proteins, a signal sequence directs proteins and is later transferred

Two-step systems

cytoplasm → periplasm = Sec/Tat

periplasm → outer membrane = Type II/Type V

One-step systems (to outside of cell)

Type I and IV

One-step systems (into another cell)

Type III and IV

Type II

Piston-like pseudopilus pushes proteins from periplasm to outer membrane; Unique to Proteobacteria; Multiple systems per cell (dedicated to particular proteins)

Type V

protein passes from periplasm through barrel structure in outer membrane; usually for proteases involved in virulence and adhesins

Type I

Multiple systems for export of different proteins; ABC (ATP binding cassette) transporter in plasma membrane component specific for a single substrate

Type IV

DNA transfer or secretion of DNA-protein complexes; pushed through pilus

Type III

Molecular syringes used to inject virulence proteins (effector proteins) into host cells (injectosome)

Type VI

Contractile; Deliver toxins to neighboring bacterial and eukaryotic cells

Constitutive gene

Always expressed because the cell always needs it. Example: housekeeping genes

Regulated gene

Usually not expressed unless needed. Example: biosynthesis genes

Inducible operon

Gene is normally off. An inducer turns it on. Example: lac (also ara)

Repressible operon

Gene is normally on. A corepressor turns it off. Example: trp

Negative control

A repressor binds DNA and blocks RNA polymerase. Example: LacI

Positive control

An activator binds DNA and helps RNA polymerase bind. Example: CAP-cAMP

Why regulate at transcription initiation?

Saves energy and materials

Helix-turn-helix (HTH) and dimer

a structural protein motifs found in DNA binding proteins, and a macromolecular complex formed by 2 protein subunits → increases affinity and specificity of binding proteins to DNA

What are the lac operon genes

lacZ: β-galactosidase, lacY: permease, lacA: transacetylase

LacI (lac repressor)

Repressor protein → blocks RNA polymerase when lactose is absent

Allolactose

Inducer- made from lactose by β-galactosidase

Binds LacI so it leaves the operator → turns transcription on

What is Leaky expression

A few lac proteins are made even without lactose. This lets a little lactose in to start induction

Function of LacZ

β-galactosidase converts lactose → glucose + galactose, also makes allolactose

Function of LacY

Lactose permease is a transport protein for lactose

Function of LacA

unknown function

CAP (CRP)

Activator - When bound to cAMP, it binds upstream of the start site, bends the DNA, and touches RNA polymerase → increases transcription rate

cAMP

Made by adenylyl cyclase → High when glucose is low

Works with CAP to increase transcription rate

Catabolite repression

Glucose is the preferred sugar. High glucose → low cAMP → lactose operons aren’t strongly induced

Diauxic growth

Two growth phases: the cell uses glucose first, pauses briefly, then switches to lactose