Central Dogma Lecture 12 Transcription II

Question 1

Rapid responses in bacteria

Answer 1

-Bacteria must respond rapidly to environmental changes
-β-galactosidase induced in ~75 seconds
-GFP induced synthetically in ~60 seconds
-Transcription and translation are coupled
-Ribosomes begin translation at 5’ end of nascent mRNA after it is extruded from RNA polymerase
-Genes transcribed at high rates have promoters that closely resemble consensus sequence
- In eukaryotes, gene expression takes hours because mRNAs must be transported to cytosol for translation

Question 2

What are operons?

Answer 2

-Co-transcribed gene clusters with a promoter plus additional sequences that function together in regulation
-Many genes in the same operon encode subunits of a larger protein complex or are involved in related processes that require coordinated regulation

Question 3

Polycistronic

Answer 3

Operon-genes are encoded on one mRNA

Question 4

What are writing conventions for genes, rna, and protein?

Answer 4

-genes: lower-case and italics (lacZ)
-RNA: lower-case (lacZ)
-Protein: capitalized, not italics (LacZ)

Question 5

What are the two conditions for the lac operon to be on?

Answer 5

-When glucose is unavailable but lactose available, cells can metabolize lactose, and lac operon is induced ~1000x
-When glucose is present, other catabolic pathways are repressed, including lac operon
-The Lac operon integrates both to avoid wasting resources

Question 6

What genes are organized in the lac operon?

Answer 6

-LacZ: encodes β-galactosidase (cleaves lactose into glucose + glucose)
-lacY: encodes galactoside permease (enables transport of lactose into the cell)
-lacA: encodes thiogalactoside transacetylase (modifies toxic glactosides to facilitate their removal)

Question 7

What does the gene lacI do?

Answer 7

-Encodes the Lac repressor
-lacI is constitutively (always) expressed

Question 8

What does the Lac repressor (LacI) do?

Answer 8

-Binds the operator sequence, which is located -7 to +28
-Prevents RNAP from initiating transcription

Question 9

What happens to turn the lac operon on?

Answer 9

-When lactose is present, bacteria metabolize a small amount of it to allolactose, an inducer
-Allolactose binds LacI, preventing it from binding DNA
-Allolactose inhibits LacI

Question 10

What is the LacI mechanism?

Answer 10

-LacI binds O1 (the main operator for lac operon) and either O2 or O3
-Lac repressor binding loops the intervening DNA to block transcription initiation

Question 11

How is the Lac operon turning on and off similar to SOS response?

Answer 11

-In absence of damage, LexA protein binds the operator (SOS box) and represses transcription of the operon
-In presence of damage, RecA destroys LexA and operator turns on
-Activation by inhibiting a negative regulator

Question 12

When is the lac operon expressed (simplified)?

Answer 12

When lactose is present and glucose is not present

Question 13

How do E. coli “sense” glucose levels?

Answer 13

-Glucose levels are transduced by Catabolite activator protein (CAP)/Crp (cyclic AMP receptor protein)
-CAP can only bind the promoter region when cyclic AMP (cAMP) is bound
-cAMP is made in E. coli when glucose is low, like “hunger” signal

Question 14

Why can’t RNAP bind the lac operon without CAP?

Answer 14

-RNAP does not bind well to the lac operon promoter because of deviations from the consensus sequence
-Therefore, needs help from CAP
-CAP only binds when cAMP is high
-lac operon is only highly transcribed when glucose is absent (high cAMP) and lactose is present

Question 15

Conditions under which lac operon is expressed (with cAMP)

Answer 15

-Glucose high, cAMP low, lactose present: low gene expression
-Glucose low, cAMP high, lactose present: high levels of gene expression

Question 16

Conditions under which lac operon is not expressed (with cAMP)

Answer 16

-Glucose high, cAMP low, lactose absent: no gene expression
-Glucose low, cAMP high, lactose absent: no gene expression

Question 17

What does the Trp operon do?

Answer 17

Regulate synthesis of tryptophan from chorismate

Question 18

How is Trp operon regulated?

Answer 18

-When tryptophan is abundant, operon should be off
-Trp repressor controls expression
-When Trp repressor binds tryptophan, Trp repressor binds to its operator to repress transcription
-As tryptophan concentration increases, tryptophan birds the Trp repressor and represses transcription (negative feedback)
-Once repression is lifted, rate of transcription is fine-tuned to cellular tryptophan requirements

Question 19

What is transcription attenuation?

Answer 19

Regulator process in which transcription is initiated normally, but is abruptly halted before operon genes are transcribed

Question 20

What is the leader region?

Answer 20

A 162 nt region at the 5’ end of the trp mRNA that controls premature termination

Question 21

What is an attenuator?

Answer 21

-A region of the leader made up of sequences 3 and 4 in (trp)
-Sequences can make different hairpins, one of which will act as an intrinsic terminator
-Involves speed of transcription and translation

Question 22

Trp operon attenuator

Answer 22

-Leader/attenuator transcript contains four segments that can form 1 or 2 sets of hairpins
-Leader mRNA is translated to form a 14 AA peptide that contains two consecultive tryptophans
-When tryptophan is abundant, an intrinsic terminator is fomed (hairpin) from segments 3 and 4
-Transcription rarely continues beyond this unless tryptophan is in short supply
-When tryptophan is scarce a different hairpin (segments 2 and 3) which permits transcription of full mRNA is formed

Question 23

Trp attenuator when tryptophan is abundant

Answer 23

-Plenty of tryptophanyl-tRNATrp
-Ribosome follows behind RNAP and blocks formation of the 2-3 hairpin because it’s on top of 2, permitting formation fo the 3-4 hairpin
-Terminates transcription

Question 24

Trp attenuator/leader when tryptophan is scarce

Answer 24

-tryptophanyl-tRNATrp is scarce, and the ribosome stalls at the tandem UGG codons
-Stalling allows for 2-3 hairpin to form as 3 is being transcribed
-Blocks formation of 3-4 hairpin long enough for RNAP to continue transcription into the remainder of the trp operon

Question 25

Other amino acid biosynthesizing operons

Answer 25

-5 are also regulated by attenuation
-Leader peptides are rich in their corresponding amino acids
-Formation of 2 structures, one of which is an intrinsic terminator

Question 26

What are riboswitches?

Answer 26

-Ligand-binding mRNA domain
-Typically found in untranslated regions at the 5’ ends
-Regulatory elements outside of coding sequence

Question 27

Riboswitch definition

Answer 27

A structured segment of mRNA that binds to a specific ligand and affects the transcription, translation, or processing of the mRNA

Question 28

How do riboswitches work?

Answer 28

-Ligand binding results in a conformational change in mRNA
-Allows for alternative mRNA structures to for +/- ligand
-RNA structures are the result of complicated base and backbone interactions
-RNA folding is analogous to protein folding

Question 29

What does the FMN Riboswitch do?

Answer 29

-Regulate transcription elongation of genes involved in FMN biosynthesis
-Only want to express when FMN is low to avoid wast

Question 30

What are some mechanisms of riboswitches?

Answer 30

-Modulate availability of intrinsic termination site
-Modulate accessibility of ribosome binding site (Shine-Dalgarno sequence)
-Modulate ability of mRNA to cleave itself