Gene Expression and Regulation in Bacteria and Eukaryotes

What was the first explicit hypothesis of what genes do, proposed by Beadle and Tatum in 1941?

Genes produce traits by determining the synthesis of proteins.

What organism did Beadle and Tatum use to prepare mutants for their experiments?

Neurospora crassa.

What is the significance of the wild-type Neurospora's ability to grow on minimal medium?

It can synthesize all the amino acids and other substances it needs.

How did Beadle and Tatum create mutations in Neurospora?

They exposed it to X-rays or UV light.

What did the arginine mutants indicate about their ability to synthesize arginine?

They could not synthesize arginine on their own and required it to be supplied in the medium.

What did the experiments by Srb and Horowitz reveal about the arginine mutants?

Different mutants had defects in different genes, supporting the one gene, one protein hypothesis.

What are the three types of arginine mutants identified by Srb and Horowitz?

arg1 mutants (grow with ornithine, citrulline, or arginine), arg2 mutants (grow with citrulline or arginine), arg3 mutants (grow only with arginine).

What is the importance of genotype in relation to phenotype?

Genotype determines phenotype.

What is the Central Dogma of molecular biology?

The flow of information is DNA → RNA → protein.

What is a codon and its significance in the genetic code?

A codon is a sequence of three nucleotides that specifies an amino acid.

What is the role of the start codon (AUG) in the genetic code?

It defines where the reading frame begins.

What are silent mutations?

Mutations that change a single base but do not result in any change to the resulting protein due to redundancy in the genetic code.

What is a missense mutation?

A mutation that causes a single amino acid change in the resulting protein.

What is a nonsense mutation?

A mutation that turns a codon that should specify an amino acid into a stop codon, resulting in a truncated protein.

What are frameshift mutations?

Insertions or deletions of bases that alter the reading frame of the mRNA, affecting all subsequent codons.

What is the role of RNA polymerase in transcription?

It reads the DNA template and synthesizes a complementary RNA strand.

What is the structure of RNA polymerase?

It is a large globular protein with several channels running through it.

What is the function of the sigma protein in bacterial transcription?

It helps RNA polymerase bind to specific sequences on the DNA template called promoters.

What is a holoenzyme in the context of transcription?

The complex of RNA polymerase and sigma that binds to the promoter.

What is the Pribnow box?

A conserved sequence (TATAAT) located 10 bases upstream from where RNA polymerase starts transcription.

What is the significance of the promoter in transcription?

It is the site on the DNA template where transcription begins.

What happens at the active site of RNA polymerase?

Phosphodiester bonds are formed during RNA synthesis.

How does the genetic code exhibit redundancy?

Multiple codons can specify the same amino acid.

What is the outcome of a frameshift mutation?

It alters the reading frame, causing all subsequent amino acids to be incorrect.

What conserved sequence occurs about 35 bases upstream of the start site in bacterial promoters?

TTGACA, known as the -35 box.

What initiates transcription in bacteria?

Transcription begins when sigma binds to the -35 and -10 boxes.

What role do sigma proteins play in bacterial transcription?

Different sigma proteins bind to different promoters, determining which genes are transcribed.

What happens after sigma binds to the promoter in bacterial transcription?

The DNA double helix opens.

How do ribonucleoside triphosphates (NTPs) contribute to mRNA synthesis?

NTPs enter a channel in RNA polymerase and are incorporated into the mRNA when complementary to the template strand.

Describe the process of transcription elongation in bacteria.

RNA polymerase moves 3' to 5' along the template DNA, unwinding the double helix, reading the DNA template, and adding complementary nucleotides to the growing RNA molecule.

What signals the termination of transcription in bacteria?

Specific sequences in the template DNA act as termination sites.

What are the three different RNA polymerases found in eukaryotic cells?

RNA polymerase I transcribes ribosomal RNAs, RNA polymerase II transcribes protein-coding genes (mRNAs), and RNA polymerase III transcribes tRNAs and other small RNAs.

What is the function of basal transcription factors in eukaryotes?

They are analogous to sigma proteins and are required to initiate transcription.

What is the TATA box and where is it located in eukaryotic promoters?

The TATA box is located 30 base pairs upstream of the transcription start site.

What are introns and exons in eukaryotic genes?

Introns are noncoding sequences that interrupt the coding regions (exons) of genes.

What is the primary transcript in eukaryotic transcription?

The first mRNA made from eukaryotic genes that contains both introns and exons.

What is the role of snRNPs in mRNA processing?

snRNPs catalyze intron splicing by forming a spliceosome that removes introns from the primary mRNA transcript.

Describe the process of intron splicing.

The intron forms a loop, the spliceosome breaks the intron at its 5' end, the ends of exons are joined, and the lariat structure of the intron is degraded.

What modifications are made to mRNA after splicing in eukaryotes?

A 5' cap of 7-methylguanylate and a poly(A) tail of 100 to 250 adenines are added.

What is the function of the 5' cap on mRNA?

It serves as a recognition signal for the translational machinery and protects the transcript from degradation.

What is the function of the poly(A) tail on mRNA?

It protects the transcript from degradation.

What is the difference between transcription and translation in bacteria and eukaryotes?

Transcription and translation can occur simultaneously in bacteria, but not in eukaryotic cells.

How does an mRNA codon specify an amino acid according to early hypotheses?

Nucleotides of a codon chemically combine with amino acid side chains through shape or charge interactions.

What did Crick's hypothesis propose about the role of adapter molecules in translation?

Adapter molecules hold amino acids in place while interacting with an mRNA codon, predicting the existence of transfer RNA (tRNA) molecules.

What are exons and introns in a gene?

Exons are the translated regions of the gene, while introns are the untranslated regions.

What is the primary transcript in eukaryotic genes?

The primary transcript is the first mRNA made from eukaryotic genes, containing both introns and exons.

Where does intron splicing occur?

Intron splicing occurs inside the nucleus.

What catalyzes the splicing of introns from mRNA?

Splicing is catalyzed by a complex of proteins and small RNAs called snRNPs (small nuclear ribonucleoproteins).

What is a spliceosome?

A spliceosome is formed when snRNPs assemble on the primary mRNA transcript to mediate intron splicing.

Describe the process of intron splicing.

The intron forms a loop, the spliceosome breaks the intron at its 5' end, the 5' end attaches to an adenine in the intron forming a lariat, the 3' end of exon 1 reacts with the 5' end of exon 2 joining them, and the lariat structure is degraded.

What modifications are made to mRNA after splicing?

Caps and tails are added to the mRNA; a 7-methylguanylate cap is added to the 5' end and a poly(A) tail of 100 to 250 adenines is added to the 3' end.

What is the function of the 5' cap on mRNA?

The 5' cap serves as a recognition signal for the translational machinery and protects the transcript from degradation.

What is the role of the poly(A) tail on mRNA?

The poly(A) tail protects the transcript from degradation.

What is the process of translation?

Translation is the conversion of a sequence of nucleotides in an mRNA to a sequence of amino acids in a protein.

Can transcription and translation occur simultaneously in eukaryotic cells?

No, transcription and translation cannot occur simultaneously in eukaryotic cells; they can occur at the same time in bacteria.

What is the Crick hypothesis regarding mRNA codons?

The Crick hypothesis suggests that adapter molecules hold amino acids in place while interacting with an mRNA codon, predicting the existence of transfer RNA (tRNA) molecules.

What is the structure of transfer RNA (tRNA)?

tRNA has a cloverleaf shape formed by hydrogen bonding between complementary bases, with a CCA sequence at the 3' end for amino acid binding and an anticodon loop that pairs with mRNA codons.

How many different mRNA codons are there, and how many tRNAs are typically present in cells?

There are 61 different mRNA codons, but only about 40 tRNAs.

What is the Wobble hypothesis?

The Wobble hypothesis states that several amino acids are specified by more than one codon, with codons for the same amino acid differing only in the third position.

What is the function of ribosomes in translation?

Ribosomes facilitate the translation of mRNA into a polypeptide chain by providing binding sites for tRNAs.

What are the two subunits of a ribosome?

Ribosomes are composed of a large subunit and a small subunit, both made of RNA molecules and proteins.

What are the three tRNA binding sites in the large ribosomal subunit?

The three tRNA binding sites are the acceptor (A) site, the peptidyl (P) site, and the exit (E) site.

What happens during translation initiation in prokaryotes?

The small (30S) ribosomal subunit binds to a sequence on mRNA just upstream from an AUG codon, which is the translation start codon specifying methionine.

What is the Shine-Dalgarno sequence?

The Shine-Dalgarno sequence is the mRNA sequence to which the 30S ribosomal subunit binds during translation initiation in prokaryotes.

What assists the binding of the 30S ribosomal subunit to mRNA during translation initiation?

Translation initiation factor proteins.

What type of tRNA binds to the AUG start codon during translation initiation?

An aminoacyl tRNA that binds N-formylmethionine (initiator methionine).

What occurs when the 50S ribosomal subunit joins the 30S subunit during translation?

It joins the mRNA and tRNAfmet.

What happens in the A site of the ribosome during translation elongation?

A new aminoacyl tRNA binds to the mRNA codon.

How is a peptide bond formed during translation elongation?

A peptide bond forms between the carboxyl end of the N-formyl methionine in the P site and the amino end of the amino acid in the A site, catalyzed by a ribozyme region of the mRNA.

Describe the process of translocation during translation elongation.

The mRNA moves through the ribosome in the 5' to 3' direction, with the tRNA in the P site moving to the E site, the peptidyl tRNA moving from the A site to the P site, and the empty tRNA being ejected from the E site.

What is required for translocation during translation elongation?

Energy in the form of GTP and assistance from elongation factors.

What is a polyribosome?

A string of ribosomes translating a single mRNA simultaneously.

What are the three stop codons in the genetic code?

UAA, UAG, and UGA.

What happens when the ribosome reaches a stop codon during translation termination?

Release factor proteins enter the A site, the completed polypeptide is released, and the ribosome separates from the mRNA.

What is transcriptional regulation in gene expression?

Control of gene expression at the level of transcription, where if a gene is not transcribed, no mRNA is made.

What is translational regulation?

Control of gene expression at the level of translation, where an mRNA is not translated due to degradation or interference with ribosome interactions.

What is post-translational regulation?

Control of gene expression where proteins are manufactured in an inactive form and must be chemically modified to become active.

How does E. coli survive in varying nutrient environments?

By switching between different food sources and efficiently utilizing available resources.

What is the significance of gene expression variability in bacteria?

Some genes are expressed constitutively while others are expressed only under specific circumstances.

What is lactose composed of?

Lactose is a disaccharide composed of glucose and galactose.

How is lactose transported into E. coli cells?

By a membrane protein.

What must happen to lactose in the cytoplasm of E. coli?

It must be broken down by a specific set of enzymes.

What is the role of ribonucleases in translational regulation?

They degrade mRNA, preventing it from being translated.

What is the most efficient control mechanism for gene expression in bacteria?

Transcriptional regulation.

What can lead to different proteins being produced from a single mRNA in both prokaryotes and eukaryotes?

Alternative start sites in prokaryotes and alternative splicing in eukaryotes.

What is the consequence of the energetic cost of gene expression in bacteria?

Not all genes are expressed at all times; expression is regulated based on environmental needs.

What is the relationship between E. coli and its intestinal environment?

E. coli competes for space and resources with numerous other bacterial species in the intestine.

What enzyme does the cell produce to break down lactose?

β-galactosidase, which breaks lactose into glucose and galactose.

What role does lactose play in the production of β-galactosidase?

Lactose acts as an inducer of the β-galactosidase gene.

What were the results of J. Monod's experiments regarding β-galactosidase production?

Cells grown on glucose only and glucose + lactose showed no β-galactosidase production, while cells grown on lactose only produced β-galactosidase.

What is the genetic locus that codes for β-galactosidase?

lacZ.

What does it mean for a cell to be LacZ-?

It means the cell is deficient in β-galactosidase enzyme.

What does it mean for a cell to be LacZ+?

It means the cell has normal β-galactosidase.

What is the genetic locus that encodes lactose permease?

lacY.

What is the significance of the lacI gene?

It encodes a repressor that negatively controls lacZ expression.

What are LacI- cells?

Mutants that do not need an inducer to activate lacZ.

What are LacI+ cells?

Wild type cells that require an inducer (lactose) to activate lacZ.

How are the genes involved in lactose metabolism regulated?

They are under both positive and negative control.

What negatively controls the expression of lacZ and lacY?

A repressor, which is the product of the lacI gene.

What is the Szilard hypothesis regarding β-galactosidase synthesis?

It states that β-galactosidase synthesis is under negative control, repressed unless lactose is available.