Genetics Exam II

What molecule was first suspected to carry hereditary information in 1923?

DNA (found in chromosomes, along with proteins and RNA).

What four molecules were initially considered candidates for hereditary material?

DNA, RNA, proteins, lipids, and carbohydrates

What did Griffith discover in 1928?

A “transforming factor” could transfer virulence from dead S bacteria to live R bacteria.

What was the key finding of Avery, MacLeod, and McCarty (1944)?

DNA is the transforming factor, not proteins or RNA.

What did Hershey and Chase demonstrate in 1952?

DNA, not protein, enters bacterial cells from viruses and directs reproduction

What are the components of a nucleotide?

Phosphate group, sugar (deoxyribose), and nitrogenous base.

What bases are purines and how many rings do they have?

Adenine and guanine; 2 rings.

What bases are pyrimidines and how many rings do they have?

Cytosine, thymine (DNA), uracil (RNA); 1 ring.

How many hydrogen bonds connect A–T and G–C pairs?

A–T: 2 bonds, G–C: 3 bonds.

What gives DNA its directionality?

Orientation of the phosphate group – synthesized 5′ → 3′.

What type of replication does DNA use?

Semiconservative – each new strand has one old and one new strand

What drives DNA synthesis energetically?

Hydrolysis of pyrophosphate (PPi) makes the reaction energetically favorable.

What are the substrates for DNA synthesis?

dATP, dTTP, dGTP, dCTP + a primer with a 3′ OH.

What experiment demonstrated bidirectional replication?

Pulse-chase experiments by Huberman and Riggs.

Where does replication start in E. coli?

oriC – a 245-bp A–T rich region with conserved 9-mer and 13-mer sequences.

What proteins initiate replication in E. coli?

DnaA (binds to 9-mers), DnaB (helicase), DnaC (helicase loader).

What keeps DNA strands from re-annealing after unwinding?

Single-stranded binding proteins (SSBs).

What relieves supercoiling in unwound DNA?

Topoisomerase

What does DNA primase do?

Synthesizes short RNA primers for DNA polymerase to begin replication.

What does DNA polymerase III do?

Main enzyme for adding nucleotides during replication; requires a primer.

What does DNA polymerase I do?

Removes RNA primers and replaces them with DNA.

What seals the DNA backbone between fragments?

DNA ligase

How is the leading strand synthesized?

Continuously in the 5′ → 3′ direction.

How is the lagging strand synthesized?

Discontinuously in short Okazaki fragments.

What is the “trombone model”?

Describes looping of the lagging strand so both strands are replicated simultaneously.

How does DNA polymerase ensure correct base pairing?

Active site geometry only fits correct base pairs; incorrect ones cause stalling.

What happens if a wrong base is added?

The strand flips into the exonuclease domain where the incorrect base is removed (proofreading).

What’s the end-replication problem?

The lagging strand cannot be fully replicated at the ends of linear chromosomes.

How is the end-replication problem solved?

Telomeres (repetitive sequences) and telomerase (adds repeats using RNA template).

Where is telomerase active?

In germ-line cells, some stem cells, and cancer cells.

Why is telomerase important?

Maintains chromosome stability and cell longevity; its loss leads to aging and cell death.

What happens in mice with a TERT gene knockout?

Telomere shortening over generations causes developmental defects and apoptosis.

What is transcription?

It’s the process of making RNA from a DNA template using RNA polymerase.

What is the central dogma of molecular biology?

DNA → RNA → Protein

Why is an intermediate (RNA) needed between DNA and protein?

DNA stays in the nucleus, but proteins are made in the cytoplasm. RNA can travel between the two.

How is RNA chemically different from DNA?

RNA uses ribose sugar and uracil (U) instead of thymine (T).

How is transcription similar to DNA replication?

Both involve polymerases, nucleotide base pairing, and synthesis of a nucleic acid strand.

How is transcription different from DNA replication?

• Uses RNA polymerase

• Makes RNA, not DNA

• Doesn’t need a primer

• Only transcribes parts of the genome

• Produces single-stranded RNA

• Less accurate and has less proofreading

• RNA is released quickly from the template

What chemical reaction occurs during transcription?

RNA polymerase adds ribonucleotides to the 3’ end, forming phosphodiester bonds.

What enzyme catalyzes RNA synthesis?

RNA polymerase

Does transcription need a primer?

No, RNA polymerase can start without one.

How many RNA polymerases are in bacteria vs. eukaryotes?

• Bacteria: 1 RNA polymerase

• Eukaryotes: 3 RNA polymerases (Pol I, II, III); focus is on Pol II

What is the sigma factor in bacteria?

A part of RNA polymerase that helps it recognize the promoter and start transcription.

What is mRNA?

Messenger RNA; it carries the message from DNA to make proteins.

What is unique about mRNA?

It’s the only RNA type that’s translated into protein.

What are tRNAs?

Transfer RNAs; they carry amino acids to the ribosome during protein synthesis.

What is rRNA?

Ribosomal RNA; it helps form the structure of ribosomes.

What are snRNAs?

Small nuclear RNAs; help process pre-mRNA in eukaryotic cells.

What are miRNAs and siRNAs?

Small RNAs that regulate gene expression after transcription.

What are ribozymes?

RNAs with enzymatic activity.

What are other types of RNA?

lncRNA, piRNA, Xist RNA, circRNA, telomerase RNA, etc.

What are the main steps of transcription?

Promoter recognition → Initiation → Elongation → Termination

What is a promoter?

A DNA sequence where RNA polymerase binds to start transcription.

What happens in initiation?

RNA polymerase unwinds DNA and starts RNA synthesis at the +1 site.

What happens during elongation?

RNA polymerase adds ribonucleotides to the 3′ end of the growing RNA strand

What are the two types of transcription termination in bacteria?

• Rho-dependent termination: Uses the Rho protein to release RNA

• Rho-independent termination: Forms a hairpin loop in RNA that causes polymerase to fall off

How can mRNA turnover be studied?

Pulse-chase experiments using radioactive uracil to track newly made RNA

Why do cells use RNA as an intermediate?

DNA stays in the nucleus, but proteins are made in the cytoplasm; RNA can move between both.

What are the three regions of a bacterial gene?

Promoter (upstream), Coding region, Termination region (downstream)

What is the +1 site?

The first nucleotide where RNA synthesis begins.

What is the bacterial RNA polymerase made of?

Core enzyme (2α, 2β, 1ω) + Sigma (σ) factor = Holoenzyme

What does the sigma (σ) factor do?

Recognizes and binds to the promoter (-10 and -35 regions)

What are consensus sequences in bacterial promoters?

Common sequences at -10 (TATAAT) and -35 (TTGACA); help RNA pol bind

What happens if the promoter deviates from consensus?

What happens if the promoter deviates from consensus?

Step 1 - Promoter Recognition

Sigma factor binds -10 and -35 regions, forming closed complex

Step 2 - Initiation

DNA "melts" to form a bubble (~18 bp); transcription begins at +1

Step 3 - Elongation

After 8–10 nucleotides, sigma detaches; RNA pol continues RNA synthesis

Step 4 - Termination (Intrinsic)

Hairpin loop + U-rich tail causes RNA pol to fall off (rho-independent)

Step 4 - Termination (Rho-dependent)

Rho binds rut site, catches RNA pol, and breaks RNA-DNA bond

How many RNA polymerases do eukaryotes have?

Three:

• Pol I: rRNA

• Pol II: mRNA, snRNA

• Pol III: tRNA, 5S rRNA

What is the TATA box?

A common eukaryotic promoter sequence (~ -25): 5′-TATAAA-3′

What are general transcription factors (GTFs)?

Proteins like TFIID, TFIIA, etc., that help RNA Pol II bind promoters

What is TFIID made of?

TBP (TATA-binding protein) + TAFs (TBP-associated factors)

What is the preinitiation complex (PIC)?

RNA Pol II + GTFs assembled at the promoter before transcription starts

What are the steps of eukaryotic transcription initiation?

1. TFIID binds TATA box
2. Other GTFs (TFIIA, TFIIB, TFIIF) and RNA Pol II join
3. TFIIE and TFIIH finish the complex
4. RNA Pol II begins transcription at +1

How is eukaryotic transcription terminated?

With a poly(A) signal + downstream termination sequence

What do enhancer sequences do?

Increase transcription by helping activator proteins loop DNA to promoter

What do silencer sequences do?

Bind repressors to block or reduce transcription

What does RNA Pol I transcribe?

rRNA genes using a core element and upstream element

What does RNA Pol III transcribe?

tRNAs, 5S rRNA using internal promoter elements (Box A/B/C)

What is pre-mRNA vs. mature mRNA?

Pre-mRNA is unprocessed; mature mRNA has been capped, polyadenylated, and spliced

What is the 5′ cap?

A methyl-guanosine added by guanylyl transferase to protect the mRNA

What is the poly-A tail?

A stretch of adenines added to the 3′ end for stability and transport

What is splicing?

Removal of introns; exons are joined to make a functional mRNA

What is the 5′ cap in mRNA?

A modified guanine nucleotide (7-methylguanosine) added to the 5′ end of pre-mRNA.

What are the 4 key functions of the 5′ cap?

1. Protects mRNA from degradation

2. Helps mRNA leave the nucleus

3. Helps remove introns

4. Helps ribosome bind and start translation

What is polyadenylation?

The addition of 20–200 adenines to the 3′ end of pre-mRNA.

What sequence signals polyadenylation?

5′-AAUAAA-3′, the polyadenylation signal, downstream of the stop codon.

What proteins help identify and cut the mRNA for polyadenylation?

• CPSF binds AAUAAA signal

• CStF binds U-rich region downstream

• CFI & CFII also help with cleavage

How is the adenine tail added?

• PAP (poly-A polymerase) adds adenines

• After the first 10, PABII proteins bind and speed up the process

What happens to the cleaved RNA fragment?

It’s bound by CFI, CFII, and CStF and then degraded.

Why must splicing be accurate?

Because translation uses 3-letter codons, even 1 wrong nucleotide causes protein errors.

How were introns discovered?

They loop out in DNA-RNA hybrids because they have no matching mRNA sequence.

What is the 5′ splice site?

Start of the intron, contains GU (nearly always).

What is the 3′ splice site?

End of the intron, contains a pyrimidine-rich region and ends with AG.

What is the branch site?

Found 20–40 nt upstream of the 3′ splice site; contains a key adenine (branch point A).

Why are these three splice sequences important?

All are required for accurate splicing to occur.

What carries out splicing?

A large RNA-protein complex called the spliceosome.

What is the spliceosome made of?

snRNPs (small nuclear ribonucleoproteins) a.k.a. “snurps.”

What happens first in splicing?

The 5′ splice site is cut and loops back to the branch point A, forming a lariat.