Exam 3 - Molecular Genetics

location of DNA in prokaryotes

nucleoid (no membrane-bound organelles)

location of DNA in eukaryotes

nucleus (separates transcription and translation)

location of ribosomes in prokaryotes

cytoplasm

location of ribosomes in eukaryotes

endoplasmic reticulum (ER) and cytoplasm

how are genes organized in prokaryotes?

in operons - close together

how are genes organized in eukaryotes?

can be far apart - no operons

protein coding content on chromosome in prokaryotes

entire gene is coding

protein coding content on chromosome in eukaryotes

introns are removed and exons are coding

pre-translation mRNA processing in prokaryotes

minimal - some trimming and phosphates may be added to 3’ end

pre-translation mRNA processing in eukaryotes

• splicing of exons

• 5’-cap (methyl group added to guanine)

• 3’-poly(A) tail

are transcription and translation coupled in prokaryotes?

yes they are

are transcription and translation coupled in eukaryotes?

no they are not

bacterial operon

clusters of co-regulated (all on or off) genes with related functions

why is there a correlation between transcription and translation in prokaryotes?

ribosomes start translating mRNA before transcription even finishes, so the rate of one process directly influences the other

What happens to transcription when translation is quick?

This ribosome movement blocks Rho-dependent termination, allowing transcription to continue (positive correlation)

What happens to transcription when translation is stalled or slow?

Naked mRNA is exposed, so Rho binds and prematurely terminates transcription (negative correlation)

In bacteria, what molecule causes premature termination when translation slows?

Rho factor

promoter

DNA sequence (100-1000 bp long) upstream of structural genes - where RNA polymerase will bind to initiate transcription

operator

region of DNA between promoter and structural genes where a repressor (regulatory protein) will bind (stops transcription)

regulatory genes

encode transcription factor proteins that influence transcription rate of the operon

repressor

turns off transcription in response to external stimuli

activator

increases transcription in response to external stimuli

What 3 regions of the promoter does RNAP recognize and bind to?

-35 region, -10 region (TATA/Pribnow box), +1 region (start site)

Which RNAP subunit interacts with the -10 and -35 promoter regions?

sigma

What happens to the sigma subunit of RNAP once transcription begins?

It falls off the core RNAP.

Beta subunit of RNAP

Has the main catalytic domain - separates DNA strands and polymerizes RNA synthesis

Beta’ subunit of RNAP

helps stabilize the DNA-RNA hybrid during transcription

Alpha subunits of RNAP

involved in assembly and stability of RNAP transcription complex

Omega subunit of RNAP

stabilizes core subunits, essential for holoenzyme assembly

Sigma subunit of RNAP

essential for promoter recognition and binding (not part of core)

Transcription - Step 1 - Initiation

• sigma subunit binds to core RNA polymerase subunits

• sigma subunit binds to promoter

• beta subunit separates DNA strands at start site, creating a transcription bubble

  • attaches first nucleotide of RNA (A or G)

Transcription - Step 2 - Elongation

• Beta subunit adds nucleotides to growing mRNA strand

• sigma subunit is displaced by RNA strand and falls off.

• Core RNAP subunits move in 5’-3’ direction

  • beta’ stabilizes DNA/RNA hybrid

  • alphas bind to upstream region of DNA (UP element at -50) to help stabilize complex

Transcription elongation complex (TEC)

• DNA enters pincers of beta and beta’

• Free nucleotides enter through beta secondary channel

• beta active site = site of polymerization

• RNA exits through beta exit channel

a? (TEC)

downstream jaws

b? (TEC)

secondary channel

c? (TEC)

active site

d? (TEC)

active site channel

e? (TEC)

RNA exit channel

f? (TEC)

beta subunit

g? (TEC)

beta’ subunt

h? (TEC)

omega subunit

i’s? (TEC)

alpha subunits

Which strand is the template strand that RNAP creates a compliment of?

the 3’-5’ strand

Which strand is the RNA product identical to with U’s replacing T’s?

the coding strand (5’-3’)

Transcription - Step 3 - Termination

• polymerization continues until RNAP encounters terminator region of DNA

• RNAP pauses after transcribing string of Us

• Inverted G-C repeats bind to each other and form hairpin

• Hairpin causes RNAP to dissociate