LC 5

What is a clone library?

A collection of bacterial colonies containing plasmids, each with different DNA inserts.

How is a clone library created?

Digest vector (plasmid) + insert with the same restriction enzymes (leave sticky ends) → combine samples →ligate inserts into vectors → transform bacteria → plate bacteria to form colonies

How can a cDNA expression library identify an unknown gene? What does the cDNA library represent?

Ligate many cDNAs into E. coli expression vectors and screen for the desired trait. The library represents actively transcribed genes from the sample of interest.

Heterogeneous

How is cDNA made and used in a cDNA expression library?

1. Combine total mRNA sample (template) with DNA primer (usually Poly-T)

2. Add dNTPs + reverse transcriptase

3. Intron-free complementary DNA (cDNA) will be synthesized from RNA template

4. Second strand cDNA synthesis with DNA polymerase

5. Ligate cDNA fragments into plasmid

6. Transform E. coli

7. Plate bacteria to form a library

If an organism has 10,000 protein-coding genes, does this mean that for a given gene, the probability of it showing up in a cDNA clone library is 1/10,000. Why or why not?

No. The probability of a gene appearing in a cDNA library depends on its expression level and the cell type or conditions sampled, not simply the total # of genes.

Highly expressed gene = lots of mRNA available → cDNA more likely to appear multiple times

Lowly expressed gene = little mRNA available → cDNA is less likely to appear

Gene not expressed in those cells = mRNA is not present => cDNA won’t appear

What are the four other kinds of plasmid libraries (excluding cDNA)?

1. gDNA (genomic DNA) for genome sequencing (in the past)

2. gDNA for screening high expression phenotypes

3. Gene variants made by in vitro mutagenesis (novel chromoprotein discovery)

4. Completely novel DNA sequences for novel functions (antimicrobial peptides-AMPS)

What are chromoproteins?

Proteins that produce color, such as RFP, GFP, and amliCP which are β-barrel

How can new amilCP colors be discovered?

Randomly mutate the amliCP gene → express mutants in E. coli → screen colonies for new colors

How was amilCP random mutagenesis conducted? What residues are involved? How does a lab produce randomization?

YG dipeptide rearranges bonds to become the chromophore during amliCP folding and maturation. Cannot be changed without abolishing color.

Surrounding CQ and S residues are targeted to randomization.

When sending a sequence to the lab, you place NNN NNN as the genetic code, and the lab provides a 1:1:1:1 ratio of the four nucleotides, which will randomly incorporate.

How is a library of amliCP mutant genes constructed?

PCR with randomized codons (using primers indicated) → recircularize with T4 DNA ligase → transform E. coli → screen colonies

*most mutations abolish color

What type of promoter is used for the amilCP mutant library?

Rhamnose-inducible promoter

It allows amliCP mutant genes to be expressed when induced by rhamnose, enabling color screening

What are antimicrobial peptides (AMPs)?

Short polypeptides (~10-50 amino acids) that kill bacteria or inhibit their growth.

How are antimicrobial peptides identified using a clone library?

Use PCR and re-circularization to insert random pepetide-encoding sequences into plasmids → transform E. coli and plate on salicylate medium.

Growth inhibition when plated identifies candidate novel AMPs under control of a salicylate-inducible promoter

How were AMP clones screened for antimicrobial activity?

1. Plate colonies on medium with no Sal. Lethal-peptide clone growths just fine

2. 8 clones plated on medium without Sal

3. Same 8 clones plated on medium with Sal → promoter induced by salicylate → AMP expressed → antimicrobials prevent own growth

What does RNA polymerase engage with? What does that determine?

Promoter DNA.

Determines directionality and stimulates initiation.

The 5’ end of the RNA is born first, and termination releases the 3’ end

What determines where bacterial transcription begins and ends?

RNA polymerase binds the promoter, including the -35 and -10 regions, to initiate transcription at the +1 transcription start site.

Transcription ends at a terminator downstream of the coding region.

What are the components of bacterial mRNA, and how do transcription and translation sites differ?

The 5’ UTR is transcribed but not translated and lies before the start codon. The coding sequence (CDS) extends from start codon to stop codon. The 3’ UTR is transcribed but not translated and lies after the stop codon.

The +1 site marks the beginning of transcription; the start codon marks the beginning of translation.

What is the structure of bacterial RNA polymerase (RNAP)?

The core enzyme consists of 2 α, 1 β, and 1 β′ subunit (α₂ββ′) and can catalyze RNA polymerization. The ω (omega) subunit is expendable.

Why can’t core bacterial RNAP efficiently initiate transcription on intact double-stranded DNA in vitro?

Core RNAP requires nicked or single-stranded DNA to initiate transcription in vitro. Because cellular DNA does not need to be nicked or single-stranded for initiation, an additional factor sigma (σ) factor is needed.

What does the RNAP holoenzyme contain?

sigma (σ) factor binds to the RNAP core, enabling it to recognize promoters, melt DNA, and initiate transcription in vivo

What is bacterial promoter, and what does it do?

A DNA segment near the transcription start site that recruits RNA polymerase and directs where and in which direction transcription begins. It also contains binding sites for regulatory proteins (activators and repressors) that modify RNA polymerase binding or activity.

What sequences are commonly found in bacterial promoters?

The -35 region often resembles TTGACA, and -10 region often resembles TATAAT. Actual promoters may differ from these consensus sequences.

What are the features and spacing of a consensus bacterial promoter?

-35 → 16-18 bp → -10 → 6-8 bp → N (+1 here)

What role does σ factor play in bacterial transcription initiation?

• σ2 binds the -10 region; σ4 binds the -35 region.

• σ factor positions RNAP at the promoter.

• Binding energy causes DNA to unwind, forming the open complex.

• RNA polymerization can then begin.

What are the two complications with transcription in bacteria?

1. A given bacterial species may have several σ factors

2. Other transcription factors

What distinguishes σ⁷⁰ from alternative σ factors in E. coli?

• σ⁷⁰ (housekeeping): Essential for life; directs RNAP to promoters for the great majority of genes.

• Alternative σ factors: Generally nonessential under normal growth conditions.

  • Recognize different promoter sequences than σ⁷⁰.

  • Redirect RNAP to specific genes needed under special circumstances.

  • Allow bacteria to respond to environmental changes, including heat, chemical, and nutritional stress.

How do activators and repressors regulate bacterial transcription, and where do they bind?

Repressors: block RNAP binding → decrease transcription

Activators: stabilize/encourage RNAP binding → increase transcription

• activators can help RNAP bind weak promoters

Sites overlapping the promoter: typically repressors

Sites just upstream of the promoter with no overlap: typically activators

Bacteria can use approximately 100-200 different regulatory proteins

What are the stages of the bacterial transcription cycle?

1. Binding

2. Closed complex (RNAP is bound, but DNA is not melted)

3. Open complex (DNA melted around +1)

4. Abortive initiation (short RNA fragments synthesized while RNAP remains at the promoter)

5. Elongation (RNAP clears promoter; σ factor no longer needed; 60 nt/sec

6. Termination (RNA synthesis ends and RNAP releases)

What is Rho, and where does it bind during rho-dependent termination? Why doesn’t Rho terminate transcription immediately?

• Rho is a homohexameric protein complex (six identical subunits)

• Binds to C-rich sites on newly synthesized RNA called rho-utilization (rut) sites

• Rho translocates along the RNA until it catches RNAP → triggering termination

• Translating ribosomes mask the RNA → preventing Rho from binding

• Rho-dependent termination is more likely when RNA is not being translated

How does rho-independent termination work?

• RNA forms a stable, G/C-rich hairpin followed by a U-rich segment

• Termination hairpin triggers RNAP to terminate transcription

• Rho is not required; the RNA itself triggers termination