BIOL 20A Midterm 2

DNA Replication

Begins at origin of replication (middle of replication fork); occurs in the nucleus

Leading strand

Continuously synthesized TOWARDS the replication fork

Lagging strand

Discontinuously synthesized AWAY from the replication fork (forming Okazaki fragments)

Transcription

DNA-directed RNA synthesis; RNA synthesized by RNA Pol; Produces a single-stranded RNA molecule complementary to one strand of the DNA double helix; occurs in the nucleus

Splicing

Can occur during/after transcription; Introns are removed to form mature RNA (line of exons); allows some genes to encode more than one RNA via alternative splicing

Translation

Ribosome moves from the 5’ to 3’ end of an mRNA as the protein is synthesized from its N to C terminus; occurs in the cytoplasm.

What level regulation is the trp operon?

Transcriptional

What level regulation is the iron/ferritin system?

Translational

Central dogma of biology

DNA —> RNA (transcription)

RNA —> Protein (translation)

Frederick Griffith

Studied pneumonia (caused by a bacterium Streptococcus pneumoniae); found that heat-treatment didn’t destroy the “transforming” activity (injected mice with living/heat-killed s/r cells)

Alfred Hershey and Martha Chase

Studied bacteriophage T2 (a virus that infects E. coli); used a waring blender and found that DNA is not protein and that it carries genetic information

James Watson and Francis Crick

DNA structure determined using X-ray crystallography

Describe the key experimental evidence that DNA is the carrier of genetic information

Hershey and Chase—The Waring Blender experiment: They labeled bacteriophages with radioactive isotopes (phosphorus-32 in DNA and sulfur-35 in protein), infected bacteria, and then used a blender to separate the phage's protein coat from the bacteria. The results showed that most of the radioactive sulfur remained outside the bacteria, while radioactive phosphorus was found within the bacteria, confirming that DNA is the primary genetic material passed on during infection. 

Describe early experimental evidence that DNA encodes proteins

1909 Archibald Garrod “Inborn Errors of Metabolism” and 1940 George Beadle and Edward Tatum (studied Neurospora strains, one gene one enzyme hypothesis)

Explain how the sequence information in DNA specifies the sequence of amino acids in a protein

The DNA sequence is interpreted in groups of three nucleotide bases (codons) which correspond to an amino acid protein

Key properties of the genetic code

Redundant, universal, and not ambiguous

Describe the sequences that determine the region of an mRNA that specify the acid sequence of the protein it encodes

Codons

Provide a rough estimate of the size of the human genome and number of genes it contains

Approx. 3000 Mb (megabase; 3 × 10^9 base pairs) of DNA; approx. 25,000 protein coding genes

Describe the role of promoters in transcription

Determines the template strand and tells RNA polymerase where to begin transcribing the DNA into mRNA

Describe what happens during transcription initiation, elongation, and termination

Initiation: RNA polymerase binds to the promoter and unwinds a short region of DNA to expose the template strand

Elongation: RNA polymerase begins synthesizing RNA complementary to the template strand (5’ end of RNA); RNA polymerase adds nucleotides complementary to the template strand to 3’ OH of RNA one at a time; RNA transcript is released as it is made, DNA double helix immediately reforms

Termination: Transcription stops when RNA polymerase reaches the termination site, then it dives to the bottom right of the DNA strand

Describe the regions of DNA that define a “gene” or “transcription unit”

Promoters and termination sites

Compare and contrast transcription by RNA polymerase and DNA replication by DNA polymerase

DNA replication by DNA polymerase:

• Uses both strands of the original DNA as a template to produce an exact duplicate of the DNA for cell division (mitosis)

Transcription by RNA pol:

• Uses one strand of DNA as a template to create an RNA molecule (which can then be translated into a protein)

Similarities:

• Both processes occur in the 5’ —> 3’ direction

• Both are catalyzed by polymerase enzymes (DNA or RNA pol)

• Both involve unwinding the DNA double helix

• Both use DNA as a template

Differences:

• In transcription, Uracil replaces Thymine

• DNA replication produces two identical DNA molecules, transcription produces an RNA molecule (mRNA, tRNA, or rRNA)

The nuclear envelope

Separates the cytoplasm from the nucleoplasm; double-membrane structure (inner and outer membrane)

Describe the three major forms of RNA processing in eukaryotic cells

Capping: addition of a modified guanine nucleotide to 5’ end of the pre-mRNA

Polyadenylation: post-transcriptional addition of 200-300 adenine nucleotides to 3’ end of the pre-mRNA

Splicing: removal of specific, internal segments (introns) of the pre-mRNA to create mature mRNA

Briefly explain how introns are recognized and removed by the spliceosome

Introns are flanked by 5’ and 3’ splice sites, signals are recognized by the spliceosome, the spliceosome removes introns and joins exons back together

Alternative splicing

Cellular process in eukaryotes; the pairing of different combinations of 5’ and 3’ splice sites; leads to multiple distinct mRNA transcripts from a single gene

Explain the roles of mRNA, tRNAs, and the ribosome in protein synthesis (translation)

mRNA: provides the codons that are later translated into a protein sequence

tRNAs: Exit site, peptidyl-tRNA binding site, and aminoacyl-tRNA binding site; recognize codons in the mRNA and link them to specific amino acids

Ribosome: the “workbench” where aminoacyl tRNAs and mRNA are brought together during translation, catalyzes formation of the peptide bond between amino acids

Explain how tRNAs match the correct amino acid with each codon in the mRNA

Aminoacyl tRNA synthetases (charging)

Describe the molecular anatomy of the ribosome, including its subunit composition and functional sites

Small subunit, large subunit, E site, P site, A site, aminoacyl tRNAs, amino acids

What happens during translational initiation, elongation, and termination

Initiation: small ribosomal subunit binds to mRNA; large ribosomal subunit completes the initiation complex

Elongation: amino acids added one at a time as polypeptide grows; codon recognition —> peptide bond formation —> translocation

Termination: when the A site encounters a stop codon, no more amino acids can be added; release factor binds the A site; covalent bond between the tRNA and the polypeptide at P site is hydrolyzed; completed polypeptide chain is released, ribosomal subunits dissociate

Silent mutations

Change nucleotide sequence, but not protein sequence; i.e., AAG to AAA (both still lysine)

Missense mutations

Change base sequence and amino acid sequence; i.e., AAG (lysine) to GAG (glutamic acid)

Nonsense mutations

Change codon to termination codon; i.e., AAG (lysine) to UAG (stop)

Effect of insertions and deletions vary depending on their…

size (will cause frameshift if not divisible by 3)

Describe the levels at which gene expression can be regulated in prokaryotic and eukaryotic cells

Prokaryotic: transcriptional level (i.e., trp biosynthesis)

Eukaryotic: multiple levels, including translational level (i.e., regulation of cellular iron levels), RNA processing, transcriptional, and stability

In general terms, describe the potential advantages of various forms of gene regulation

Saves energy and resources, allows flexibility, enables rapid cellular responses to environmental changes, and drives cellular differentiation and development

With reference to the trp operon, explain the mechanism by which E. coli bacteria regulate the levels of the enzymes involved in tryptophan biosynthesis

The trp repressor is a regulatory protein that binds the trp operator only in the presence of tryptophan; low trp = repressor doesn’t bind, transcription occurs & high trp = repressor binds operator; transcription is repressed

Explain the mechanisms used by eukaryotic cells to regulate the levels of ferritin in response to changes in cellular iron levels

Iron response proteins (IRPs) bind iron-response elements (IREs) when iron levels are low (ferritin levels drop because translation of ferritin mRNA is blocked); when iron levels are high iron binds IRP and IRPs do not bind IREs (ferritin levels increase, translation of mRNA is enabled)

Explain the importance of sequence-specific DNA and RNA-binding proteins in the regulation of gene expression, with references to members of each class of regulatory protein and the sequences to which they bind

They regulate the gene expression of different cellular components based on the demands of the cell in response to environmental or physiological signals

ex:

• trp repressor protein; binding/not binding to trp operator for transcription of trp operon to produce trp

• IRP binding/not binding to the IRE in mRNAs for translation of ferritin mRNA to produce ferritin