Gene expression and transcription

gene expression

info encoded in genes direct biosynthesis of proteins

central dogma of molecular bio

field born in 1957- Crick and Watsons

reverse transcriptase

An enzyme encoded by some certain viruses (retroviruses) that uses RNA as a template for DNA synthesis.

gene expression process is

conserved in microbes but signifcant structure differences

gene expression dif in domains

transcription

DNA to RNA

RNAP in archaea and eukarya have many

auxillary subunits (GTFs)

RNAP in prokaryotes vs eukarya

prokarotes have 1 RNAP

eukarya have 3 RNAP I-III

RNAP in bacteria was first described in

rats-1995

E coli by chamberlain- 1962

archaea-1971

RNAP in bacteria is made of

5+1 subunits (400 kDa)

two alpha factors, one beta factor, one beta prime factor, one omega factor

+sigma factor

sigma subunit

used for initiation and is dissociable

directs RNAP to correct promoter of gene by binding to the -10 and -35 box

beta factor

150 kDa

active site, DNA binding

beta prime factor

150 kDa

bit bigger than beta

DNA binding and stability

alpha factors

36 kDa each

assembly, regulation

omega factor

10 kDa conformation

promoters

noncoding DNA sequences provide instructions to RNAp and other molecules to control transcription

promoters exist in

all microbial domains

promoters control

how genes turn on and hwo gene is expressed

promoters contain

1. RNAP binding site (-10=pribnow box in bacteria and TATA in euk. and arch. and -35 boxes)

2. spacer

3. initiation site (+1): where first RNA nucleotide is placed

recombinant RNAP used in

IVT to generate vaccines/other therapies

transcription initiation

RNAP binds to boxes of gene promoter

strand separation to transcription bubble at promoter (OC)

RNAP begins synthesis of RNA, sigma factor dissociates and intitation complete

eukaryotes and archaea use what instead of sigma factors of OC formation?

GTFs

most bacteria have how many sigma factors

greater than 1

E coli have 7

S cellolosum >100

antimicrobials can target

sigma factors-sigmacidin

B-rifamycin

B prime- fibaxomycin

transcription elongation

core RNAP goes to TEC

synthesis of ssRNA from DNA template

about 1 kbp in prok. and 20 kbp in eukary.

transcription termination

in prokaryotes is either Rho dependent or intrinsic/Rho-independent

intrinsic termination

Rho independent

requires:

1. pause site (poly U)

2. hairpin loop (transient dsRNA species formed by complimentary base bairing between tandem repeats)

3. N-utilization substance protein (NusA)- about 60-80% genes- binds to RNA to form HP loop proximal to RNAP

intrinsic termination what leads to mRNA release

contact bet RNAP, HP, and NusA leads to dissolution of RNA:DNA duplex at poly-U, pulls apart

Rho dependent termination requires

1. rho

2. rho utilization (rut) site/box

3. pause site (about 20-30% of genes)

rho dependent termination includes process of

RNA translocation

Rho catches up to stalled RNAP for termination about 40 bp/sec

rho in bacteria and archaea

rho-like proteins

FttA and EtA

for transcription termination eukarya use

poly-A signal sequences (about 90% mRNAs) and unique termination proteins

biocydomycin

peptide antibiotic inhibits Rho-dependent termination in bacteria