Micro Exam Three

In reverse transcription polymerase chain reaction (RT-PCR), an RNA population is converted to ______ by reverse transcription (RT), and then the —— is amplified by the polymerase chain reaction (PCR)

cDNA (complementary DNA)

In RT-PCR what enzyme converts RNA to cDNA?

reverse transcriptase

Primers (gene-specific oligonucleotides)

• Short strands of DNA.

• They tell the DNA polymerase where to begin copying the target DNA.

• Help ensure the PCR amplifies the gene scientists want to study.

RNase inhibitor

• Protects RNA from being broken down by enzymes called RNases.

• RNases naturally break down RNA.

• Prevents the RNA sample from being destroyed before it can be converted into cDNA.

Housekeeping genes

genes that are constantly expressed in cells because they perform basic functions necessary for cell survival and normal activity

In bacteria, all transcription is performed by a ______ type of RNA polymerase. This polymerase contains four catalytic subunits and a single regulatory subunit known as _______ .

single, sigma

In eukaryotes, transcription is achieved by _______ different types of RNA polymerase (_______)

three, RNA pol I-III

RNA pol I transcribes _________________

ribosomal RNAs (rRNAs)

RNA pol II transcribes RNAs that will become ________________ and also ______________.

messenger RNAs (mRNAs), small regulatory RNAs

RNA pol III transcribes small RNAs such as _______________

transfer RNAs (tRNAs)

Stages of Transcription

1. Initiation

2. Elongation

3. Termination

RNA uses uracil (U) instead of _________

thymine (T).

DNA → RNA base pairing:

• A (adenine) → U (Uracil)

• T (thymine) → A (adenine)

• C (cytosine) → G (guanine)

• G (guanine) → C (cytosine)

Regulatory proteins control whether _________________.

genes are turned ON or OFF.

What are the two types of regulatory proteins?

Activator and Repressor

Regulatory proteins bind to specific __________ to control gene expression.

DNA sequences

The lac operon is a group of bacterial genes that allows bacteria, such as E. coli, to break down _________.

lactose.

The lac operon is normally _____ but can be turned ______ when lactose is available.

OFF, ON

When lactose is absent…

• Repressor binds to the operator.

• RNA polymerase is blocked.

• Genes are OFF.

When lactose is present…

• Some lactose is converted into allolactose.

• Allolactose binds to the repressor.

• Repressor changes shape and releases the operator.

• RNA polymerase can transcribe the genes.

lacZ

produces beta-galactosidase, which breaks down lactose.

lacY

produces lactose permease, which helps lactose enter the cell.

lacA

produces transacetylase, which transfers acetyl groups.

The lac operon is controlled by TWO different regulatory mechanisms known as what?

• NEGATIVE REGULATION: controlled by the repressor.

• POSITIVE REGULATION: controlled by CAP and cAMP.

_____ and _____ work together to help turn the lac operon ON, especially when glucose is LOW.

CAP, cAMP

CAP (Catabolite Activator Protein)

a regulatory protein that helps RNA polymerase bind to DNA and increase transcription.

cAMP (Cyclic AMP)

cAMP is a small signaling molecule inside the bacterial cell. cAMP binds to CAP and activates it.

When glucose is LOW, cAMP levels _______

increase

When glucose is HIGH, cAMP levels ________.

decrease

The lac operon has its HIGHEST activity when lactose is ______ AND glucose is _____.

present, absent

The trp (tryptophan) operon works _________ to the lac operon.

opposite

The trp operon is a repressible operon, meaning it is normally ___ but can be turned _____.

ON, OFF

Purpose of the trp operon

to help bacteria produce the amino acid tryptophan

When tryptophan is LOW:

• Repressor is inactive.

• Cannot bind to the operator.

• Genes are transcribed.

• Bacteria make tryptophan.

When tryptophan is HIGH:

• Tryptophan binds to the repressor.

• Tryptophan acts as a corepressor.

• Repressor becomes active.

• Repressor binds to the operator.

• Transcription stops.

Purpose of the Polymerase Chain Reaction (PCR)

to make millions of copies of a specific DNA sequence.

Steps of the PCR

1. Denaturation

2. Annealing

3. Extension

What happens in the denaturation step of the PCR?

• Heat separates the double-stranded DNA into two single strands.

• Hydrogen bonds break.

What happens in the annealing step of the PCR?

• Temperature decreases.

• Primers attach to complementary DNA sequences.

• Primers identify the region being copied.

What happens in the extension step of the PCR?

• Taq polymerase adds DNA nucleotides.

• New complementary DNA strands are created.

dNTPs (deoxyribonucleotide triphosphates.)

They are the building blocks of DNA used during DNA replication and PCR.

Purpose of DNA sequencing (Sanger sequencing)

to determine the exact order of nucleotides in a DNA strand.

The four DNA bases of DNA sequencing:

• Adenine (A)

• Thymine (T)

• Cytosine (C)

• Guanine (G)

What does ddNTP do?

terminates DNA syntheisis

Why does ddNTP stop DNA synthesis?

Because it lacks the 3'-OH group needed to attach the next nucleotide.

What happens when tryptophan levels are HIGH?

Tryptophan acts as a corepressor, activating the repressor so it can bind the operator. So, the trp operon turns OFF.

What makes ddNTPs different from regular dNTPs?

ddNTPs lack a 3'-OH group, so another nucleotide cannot be attached.