CH 7 & 8 - Prokaryotic Genomes and Gene Expression

A phenotype generally results from the functioning of one or more:

Genes

• can have genotype but wont see phenotype all the time

DNA —→RNA—→Protein

Central Dogma

Transcription

DNA—→RNA

• RNA polymerase; mRNA

Translation

RNA—→Protein

• genetic code, transfer RNA’s; ribosomal RNA’s

Transcription and translation are ______ in prokaryotes

coupled

Sense strand

coding strand

• mRNA = “sense” strand (almost) —→ due to uracil

Antisense strand

non-coding template strand

Compare sense mRNA to sense DNA strand are the same except _______

for the T/U substitutions

Genetic Code

• redundant: 64 codons (20 amino acids)

• start/stop codons

• represents mRNA

Codons process

• The “A” in AUG start codon is not the first base in the transcript

Prokaryotic Genomes

• Genome

• Transcriptome

• Proteome

Genome

the totality of DNA in a cell

• chromosome and plasmids

• PROKARYOTES are HAPLOID (1 chromosome)

Transcriptome

total transcripts present at a given time

• transcripts DISAPPEAR QUICKLY

Proteome

all the types and numbers of proteins expressed at a given time

Prokaryotic genome organization

• Most possess a single circular chromosome

• Size: 500,000 to 9,000,000 bp (order of magnitude < eukaryotes)

• May also possess small, circular, extra-chromosomal segments called plasmids (1000 – 25,000 bp)

operon vs. regulon

operon - single regulatory sequence

regulon - a collection of operons

Cistron

gene

• Monocistronic: one prmoter, one gene

• Polycistronic: one promoter, multiple genes

Operon basic structure

promoter

defines the start of the gene (common to prokaryotes and eukaryotes)

Eukaryotic Gene Structure (for comparison)

Operon gene structure

Operon: promoter + operator + structural genes

• Regulatory gene is upstream

• Regulatory gene —→ regulatory protein —→ binds to operator and prevents transcription

• If regulatory gene is inactive: structural genes —→ transcription into polycistronic message —→ translation into enzymes (proteins) which guide metabolic pathways

Sigma factors

interacting w/different promoters; sigma factors guide RNA polymerase to the promoters

Regulon

controls multiple operons

• collectively control operons common to particular metabolism via specific sigma factor

Plasmids

small, extra-chromosomal, circular DNA segments 2 – 25 kbp in size.

• Plasmids NOT necessary for survival

• Autonomous; possess an ori

• Some can integrate into chromosome

• Some are transferable

How do low copy number plasmids and high copy number plasmids affect the latter?

High copy number plasmids provide higher gene expression and protein yields, but can lead to a metabolic burden, protein aggregation, and lower cell growth. Conversely, low copy number plasmids are less of a burden and are better for expressing toxic proteins, studying genes at a more physiological level, or maintaining a more stable phenotype. 

R factor, catabolic plasmids, F factor

resistance to antibiotics, catabolic pathway, fertility (transfer of plasmids - conjugation)

Bidirectional replication (plasmids)

same as chromosomal replication

Rolling circle replication

unique to plasmids

• Nick formation (RepA); exposes 3’- OH of nucleotide

• Nick made by RepA at ori extended by RNA polymerase

• Rep releases old (+) strand and new cell recieves copy of plasmid

• synthesis of complementary (-) strand for plasmid in new cell

Plasmid Inheritance

Plasmids do not carry essential* genes how are they maintained in the host cell?

• Possess plasmid genes that benefit the host under certain conditions *antibiotic resistance (Selective pressure)

• Integrate plasmid into the chromosome

• High/Low copy # of plasmids

High copy # plasmids

Low copy # plasmids

• possess a partitioning system (par proteins)

• Par proteins - guide plasmid to opposite end (requires energy); serve to segregate and partition plasmid and its copy

Bacterial RNA Polymerase

Bacterial RNA polymerase “holoenzyme”: a core polymerase that synthesizes mRNA : has 4 subunits: 2 alpha (α)subunits, 1 β, and 1 β’

• Sigma factor guides RNA polymerase to the promoter, then unbinds from core polymerase

-35 and -10 bp

Most sigma factors recognize promoter sequences at positions -35 bp, -10 bp upstream of transcription start.

Consensus sequences are conserved sequences

rich in T, A bonds b/c it has 2 DB compared to 3 in G, C (less energy to break)

σ70 sigma factor

common for most bacterial genes

Changes in promoter sequence:

up (increase activity) or down (decrease activity)

Promoter strength:

strong (high expression) v. weak (low expression)

• strong vs weak binding (weak promoters are NOT BAD, they just code for less genes)

Ribosome (70S)

large subunit (50S) + small subunit (30S)

• 30 S includes —→ 16 S - rRNA and proteins

Shine-Delgarno sequence:

ribosome binding site; binds to 16S ribosomal RNA of small (30S) ribosomal subunit;ribosome binding followed by translation of transcript toform polypeptide