Bio 2960 Exam 3

Gene Structure and Genomes: Bacterial protein-coding genes include what components?

Promoter, transcription start site (TSS), regulatory sequences, transcription terminator, open reading frame (ORF), start codon, stop codon, and 5’ and 3’ untranslated regions (UTRs)

What is an operon?

A cluster of genes transcribed together under a single promoter into one polycistronic mRNA

Introduction to Transcription: What is spatial orientation of DNA?

The positional relationship of DNA elements using terms such as proximal, distal, upstream, and downstream

What do proximal and distal mean in DNA orientation?

Proximal means close to the gene; distal means farther away

What do upstream and downstream mean in transcription?

Upstream is toward the 5’ end before the TSS; downstream is toward the 3’ end after the TSS

What are the key components in transcription?

DNA, mRNA, RNA polymerase, ribonucleic acid, template strand, non-template (coding) strand, and DNA unwinding/rewinding

What is the template strand?

The DNA strand used by RNA polymerase to synthesize RNA

What is the non-template strand?

The coding strand, which matches the RNA sequence except T is replaced with U

What are the stages of transcription?

Initiation, elongation, and termination

What happens during DNA unwinding and rewinding in transcription?

DNA opens to form a transcription bubble and re-anneals after RNA polymerase passes

Bacterial Transcription Initiation: What is the promoter?

A DNA sequence that recruits RNA polymerase and defines the transcription start site

What are the -10 and -35 regions?

Consensus promoter sequences recognized by sigma factor for RNA polymerase binding

What is a consensus sequence?

The most common nucleotide sequence found at a functional DNA site

What is the transcription start site (+1)?

The first nucleotide transcribed into RNA

What is RNA polymerase in bacteria?

The enzyme that synthesizes RNA from a DNA template

What is the sigma (σ) subunit?

A protein that helps RNA polymerase recognize promoters

What is the transcription bubble?

The region of unwound DNA where transcription occurs

Bacterial Transcription Elongation: What is the direction of RNA synthesis?

RNA is synthesized 5’ → 3’

What is an RNA-DNA hybrid?

The temporary pairing between nascent RNA and template DNA

What is a nascent transcript?

The newly forming RNA molecule

What is the role of the 3’ OH in transcription?

It provides the site for addition of incoming nucleotides

What is rNTP hydrolysis used for?

It provides energy for RNA chain elongation

Bacterial Transcription Termination: What is intrinsic termination?

Termination caused by formation of a GC-rich hairpin followed by U-rich sequence

What is a hairpin loop?

A secondary RNA structure that destabilizes transcription

What is rho-dependent termination?

Termination requiring rho helicase to release RNA from DNA

What is a rut site?

RNA sequence where rho protein binds to initiate termination

Describe how genetic information within a cell is organized.

Genetic information is organized into genes, operons, and genomes with regulatory and coding regions controlling transcription and expression

Define and diagram a prokaryotic gene; identify the role of each component part.

A prokaryotic gene includes promoter (initiation), TSS (+1), ORF (protein coding), start/stop codons, UTRs (regulation), and terminator (end transcription)

Within a genome, genes can be oriented in different directions. What does this mean?

Genes can be located on either DNA strand and transcribed in opposite directions

Not all genes encode proteins. What are some gene products?

Some genes produce functional RNA such as rRNA, tRNA, or regulatory RNAs

Compare and contrast a prokaryotic gene and operon.

A gene encodes one product; an operon is multiple genes under one promoter producing a single polycistronic mRNA

How can ORFs be interpreted in genome diagrams?

ORFs are represented as arrows showing direction of transcription and translation

How are ORFs described relative to each other?

Using upstream, downstream, proximal, distal, and strand orientation

Can ORF orientation determine if genes are in the same operon?

Yes; same direction and proximity suggest possible operon membership

Outline the steps of bacterial transcription.

Initiation (promoter binding), elongation (RNA synthesis), termination (intrinsic or rho-dependent)

How do you determine RNA sequence from DNA?

RNA matches coding strand except T is replaced with U; complementary to template strand

What is the role of the promoter?

It recruits RNA polymerase and positions the transcription start site

In a transcription bubble, what is the template strand?

The strand read by RNA polymerase to synthesize RNA

In a transcription bubble, what is the direction of RNA polymerase movement?

Moves along template strand 3’ → 5’

What is the directionality of RNA synthesis?

RNA is synthesized 5’ → 3’

Where is the promoter relative to the transcription bubble?

Located upstream of the bubble

Compare rho-independent and rho-dependent termination.

Rho-independent uses hairpin + U-rich sequence; rho-dependent uses rho helicase to detach RNA from DNA

Basal expression; Negative regulation, Positive regulation

Basal expression is the default level of gene transcription; negative regulation involves repression of transcription; positive regulation involves activation of transcription

Promoter (P), RNA polymerase, operator (O), Repressor, activator binding site, activator, inducers (allosteric effectors)

Promoter recruits RNA polymerase, operator is repressor binding site, repressor blocks transcription, activator binding site recruits activator, activators enhance transcription, inducers are molecules that modify regulatory proteins allosterically

Example: Lac operon

A bacterial operon that regulates lactose metabolism using inducible negative regulation and positive regulation via CAP-cAMP

Polycistronic (multi-ORF) mRNA, lactose, glucose, galactose, lacZ, beta-galactosidase, lacY, permease, lacI, Repressor

Polycistronic mRNA encodes multiple proteins (lacZ, lacY, lacA); lacZ encodes beta-galactosidase, lacY encodes permease, lacI encodes the repressor; lactose induces the system; glucose inhibits expression indirectly

lac operon mutants: lacY-, lacZ- (ΔZ), lacP, lacOC, plasmid to introduce extra copy

lacY-: no permease; lacZ-: no beta-galactosidase; lacP: promoter defect blocks transcription; lacOC: constitutive expression; plasmid (F’) introduces extra operon copy in trans

“cis” and “trans” acting regulatory factors

Cis-acting elements are DNA sequences affecting only linked genes; trans-acting factors are diffusible proteins or RNAs that act on multiple DNA targets

catabolite “repression”: glucose, positive regulation, catabolite activator protein (CAP), cAMP, CAP binding site on DNA

Low glucose increases cAMP, activating CAP which binds DNA and enhances transcription; high glucose lowers cAMP leading to catabolite repression

DNA binding proteins

Proteins that recognize specific DNA sequences through structural motifs and hydrogen bonding in the major groove

Dimer of DNA binding proteins, major groove, alpha helix

Many DNA-binding proteins function as dimers; alpha helices insert into the major groove to read base-specific patterns

Genetic Code: Triplet code, codon, redundancy, synonymous codons, start codon, stop codons, possible reading frames, Open Reading Frame (ORF), inosine, wobble pairing

The genetic code uses three-base codons; multiple codons can encode the same amino acid (redundancy); start codon (AUG) initiates translation; stop codons terminate translation; reading frames determine codon grouping; wobble pairing and inosine allow flexible tRNA recognition

mRNA, codon, tRNA, anticodon, polysome

mRNA carries codons; tRNA carries anticodons and amino acids; polysomes are multiple ribosomes translating one mRNA simultaneously

Describe the processes that activate or repress transcription in bacteria using the lac operon as an example.

The lac operon is regulated by negative control (lac repressor binding operator) and positive control (CAP-cAMP activation), allowing transcription only when lactose is present and glucose is low

Contrast positive and negative transcriptional regulation.

Negative regulation uses repressors to block transcription; positive regulation uses activators to enhance transcription

Diagram the lac operon, including both regulatory and protein-coding regions of the DNA (include: lacI, promoter, operator, lacZ, lacY, lacA, CAP binding site).

lacI encodes repressor; promoter recruits RNA polymerase; operator binds repressor; CAP binding site binds CAP-cAMP; lacZ, lacY, lacA encode lactose metabolism enzymes

Add to your diagram which proteins bind to the regulatory regions (include: Repressor, RNA Polymerase, CAP).

Repressor binds operator; RNA polymerase binds promoter; CAP binds CAP binding site when complexed with cAMP

Predict the types of interactions that allow proteins to bind to a specific sequence of DNA.

Hydrogen bonding, ionic interactions, and shape complementarity in the major groove allow sequence-specific DNA binding

Add to your diagram the name and function of the proteins encoded by lacZ and lacY genes.

lacZ encodes beta-galactosidase which breaks down lactose; lacY encodes permease which transports lactose into the cell

Predict whether various lac operon mutants can produce functional beta-galactosidase (lacZ) or permease (lacY) under different growth conditions.

lacY- cannot transport lactose; lacZ- cannot metabolize lactose; lacP prevents transcription; lacOC causes constitutive expression

Predict the outcome of combining chromosomal mutations with an extra copy of lac operon in trans.

Trans-acting lacI can complement some mutations; cis mutations like lacP and lacOC cannot be rescued by extra copies

Predict whether beta-galactosidase (lacZ) and permease (lacY) expression will be higher or lower if glucose is added to the media. Explain in terms of CAP.

Expression is lower because glucose reduces cAMP levels, preventing CAP activation and decreasing transcription

Apply these principles of transcription regulation to a new example of a bacterial operon.

Operons integrate environmental signals using repressors and activators to regulate gene expression

When given a description, determine whether a factor is acting in "cis" or in "trans" and be able to explain your choice.

Cis elements are DNA sequences on the same molecule; trans factors are diffusible proteins or RNAs that act on multiple targets

Appreciate that the genetic code that relates mRNA sequence (codons) to protein sequence (amino acids) is nearly universal and partially redundant.

The genetic code is nearly universal and redundant, meaning multiple codons can encode the same amino acid

Infer the amino acid sequence with directionality (N- to C-) translated from a given mRNA sequence, when provided with a genetic code table.

Ribosomes read mRNA 5’→3’ and synthesize proteins from N-terminus to C-terminus

Explain why there are 6 possible reading frames for a region of double stranded DNA.

Each strand has three reading frames, and two strands give a total of six frames

Determine the most likely reading frame for a region of DNA.

The correct reading frame is the one with a continuous ORF from start codon to stop codon

Understand the basic role(s) of the following RNAs during translation: tRNA, rRNA, mRNA

mRNA provides codons; tRNA delivers amino acids; rRNA catalyzes peptide bond formation

Determine which factors “know” the genetic code

tRNAs and aminoacyl-tRNA synthetases determine codon-to-amino acid matching during translation

Genetic code: Triplet code, start codon, stop codons, Open Reading Frame (ORF)

The genetic code uses triplet codons; ORFs begin at a start codon (AUG) and end at stop codons (UAA, UAG, UGA)

Key structures: mRNA, codon, tRNA, anticodon, polysome

mRNA carries codons; tRNA carries anticodons and amino acids; polysomes are multiple ribosomes translating one mRNA simultaneously

Ribosome: rRNA, 16S rRNA, protein, small (30S) subunit, large (50S) subunit, A (aminoacyl), P (peptidyl) and E (exit) sites

The ribosome is made of rRNA and proteins; the 30S subunit contains 16S rRNA; the 50S is the large subunit; A site binds incoming tRNA, P site holds growing peptide, E site is exit

tRNA Charging: Aminoacyl-tRNA synthetases, substrates: ATP, amino acid, tRNA; product: charged tRNA

Aminoacyl-tRNA synthetases use ATP to attach the correct amino acid to its corresponding tRNA, producing a charged tRNA

Initiation/Ribosome Assembly: fMet (formylmethionine), initiator tRNA, start codon (AUG), Shine Dalgarno sequence (= Ribosome Binding Site of mRNA), Initiation factor IF2*, GTPase, 30S initiation complex, 70S initiation complex

The Shine-Dalgarno sequence aligns the 30S subunit; IF2 delivers fMet-tRNA to the AUG in the P site; GTP hydrolysis drives formation of the 70S initiation complex

Elongation: peptide bond formation, ribozyme, ribosome translocation, Elongation factors EF-Tu*, EF-G, GTPase, ribosome A, P, E sites

Peptide bonds are formed by rRNA (ribozyme activity); EF-Tu delivers charged tRNA to the A site; EF-G drives ribosome translocation using GTP hydrolysis

Termination: Release factor RF, molecular mimicry

Release factors mimic tRNA shape and bind stop codons to trigger polypeptide release

Differences from Prokaryotic Transcription: spatial separation of transcription and translation; transcript processing; general transcription factors; cis-acting elements; “ground state” of transcription, promoter composition

Eukaryotes separate transcription (nucleus) and translation (cytoplasm), require RNA processing and general transcription factors, and rely on complex promoter and regulatory elements with a more “off” default state

Gene Structure in Eukaryotes: promoter, core promoter, enhancer (distal, proximal, downstream), intron, exon

Eukaryotic genes include promoters, a core promoter (e.g., TATA box), enhancers (distal/proximal/downstream), introns, and exons that define regulatory and coding regions

DNA Packaging in Eukaryotes: nucleosome, histone, “beads on a string” (10 nm fiber); chromatin (30 nm fiber); heterochromatin; chromosome

DNA is wrapped around histones forming nucleosomes (“beads on a string”), which compact into chromatin fibers and further into chromosomes; heterochromatin is highly condensed and transcriptionally inactive

Nucleosome structure: histone octamer, histone-histone “handshake” interaction, histone-DNA interaction, minor groove, core histone domain, histone tail domain, modifications to tail: lysine acetylation, methylation

DNA wraps around a histone octamer via histone-DNA interactions; histone tails protrude and can be modified by acetylation or methylation to regulate chromatin state

In a test tube (i.e. naked DNA): core promoter, TATA box; pre-initiation complex, general transcription factors: TFIID (and TBP within it), TFIIB, and TFIIH (helicase and kinase), RNA Polymerase II C-terminal domain (CTD), open complex

TFIID binds the TATA box, recruiting TFIIB and TFIIH; TFIIH unwinds DNA and phosphorylates RNA Pol II CTD to form the open complex