3L4-5 Prokaryotic Transcription

mRNA

Messenger RNA (mRNA) contains sequence of bases that encodes the primary amino acid sequence for a protein.

A mRNA serves as the template for translation by a ribosome.

tRNA

Transfer RNA (tRNA) carries an amino acid into the catalytic site of a ribosome.

The tRNA base pairs to mRNA to ensure the selection of the correct amino acid for incorporation into a nascent polypeptide chain.

rRNA

Ribosomal RNAs (rRNA) are structural components of a ribosome, the enzyme that catalyzes translation.

ncRNA

Noncoding RNAs (ncRNAs) do not encode proteins but have a variety of catalytic, structural, and regulatory functions.

gene

DNA encoding a protein + DNA regulatory elements

primary transcript

bacteria = DNA > primary transcript / mRNA = ready for translation

eukaryote = DNA > primary transcript > modified into final mRNA = ready for translation

ORF

open reading frame, start codon > end codon

continuous sequence that encodes primary sequence of a protein

“cistron” or “coding sequence”

cis v trans acting factors

trans-acting factors = diffusible, DNA-binding proteins (TFs)

cis-acting factors = fixed places tied to genes, only impact their gene

transcription factors

DNA-binding proteins, trans-acting factors

transcription is catalyzed by

RNA polymerase

transcription starts at ___, ends at ___.

promoter (+1);

terminator

regulatory elements are located

upstream

proximal v distal

close;

far

RNA polymerase mechanism

similar to DNA polymerase

• RNA polymerase can start synthesizing RNA de novo (without a primer )

• Substrate is ribonucleoside 5’triphosphates (NTP)

• Forms phosphodiester bond between 2 NTPs to begin RNA synthesis

• The 5’ end will contain 3 phosphate groups

• requires template DNA

• no proofreading exonuclease activity

template v nontemplate

noncoding v coding

promoter sequence defines

template v nontemplate

prokaryotic RNA polymerase

e coli has only one

core enzyme and holoenzyme

RNA polymerase core enzyme

• carries out transcription elongation

• subunits a2 are essential for enzyme assembly and interact with activators

• Subunits β and β’ form the catalytic core

• Subunit ω provides structural stability

RNA polymerase holoenzyme

initiates transcription and synthesis of first 10 NTs

• subunit sigma recognizes a promotes

stages of transcription

Initiation

• Rate of transcription initiation is the major determinant of gene expression

• Separate DNA strands

• RNA polymerase holoenzyme

Elongation

• RNA polymerase core enzyme

• Synthesizes RNA 5’→3’

• Moves along template strand 3’→5’

Termination

• RNA polymerase core enzyme reaches terminator sequence to stop transcription

prokaryotic promoters

located on -35 and -10 regions;

RNA poly sigma subunit recognizes and binds, bending DNA to open it

consensus sequence

certain NTs are very common at each position

sigma’s consensus sequences

-35 = 5 TTGACA 3

-10 = 5 TATAAT 3

UP

upstream promoter element;

third AT rich recognition element, -40 > -60, highly expressed genes

bound by alpha subunit of RNA polymerase

constitutive promoters

active always, used in housekeeping genes

rate of transcription initiation is determined by __

similarity to bacterial promoter consensus sequence

strong v weak promoters

high sequence similarity with consensus;

several base differences

transcription initiation

1. holoenzyme binds to promoter = closed complex

2. unwinding DNA 12-15 bp > transcription bubble = open complex

3. holoenzyme initiates RNA synthesis and synthesizes 10 NTs

closed complex

holoenzyme + promoter

sigma 70 identifies promoter and makes protein-DNA interactions

transcription elongation

1. sigma70 dissociates > core enzyme

   1. RNA polymerase completes promoter clearance

   2. NusA protein replaces sigma70

2. transcription elongation rate increases

3. translation begins before transcription finishes

   1. NusG binds RNA poly and ribosomes link complexes

transcription elongation rate can be slowed by

RNA secondary structures

NusG

binds RNA poly and ribosomes link two complexes

rho (p) independent termination

terminators have hairpin region in RNA

long repeat of U, DNA has lots of A (U-A = 2 H bonds, weak)

rho dependent termination

rho-dependent terminators have CA-rich sequence called rut (rho utilization) element

rho binds the rut in RNA, promotes release of RNA

rho

protein factor with ATP dependent RNA-DNA helicase

rut site

Rho UTilization site, rho binds to and promotes release of RNA

operon

coordinately regulated gene clusters, arranged as transcription unit;

some have constitutive promtoers

polycistronic mRNA

formed with one promoter and terminator,

has multiple ORFs encoding a different protein

inducible promoters

gene regulation changes to environment, it can be turned on and off

specificity factors

alter specificity of RNA poly for given promoter / set of promoters

repressors v activators

enhance RNA polymerase-promoter interaction

transcription factors are ___acting elements

trans

predominant structure in e.coli is

helix turn helix motif

recognition alpha helix

makes sequence-specific interactions

motif causes alpha helix to stick out from protein,

interacts with bps through grooves, forms H bonds and VDW with bps

exposed base pair chemical groups

each base pair presents a unique set of chemical groups in the major groove

major groove v minor groove specificity

major can always specify AT v TA v GC v CG

minor groove can only show AT/TA v GC/CG

helix-turn-helix motif

predominant DNA binding domain, defined by 2 alpha helixes.

second helix is the recognition helix

bacterial TFs

most bind DNA with dimers (2 subunits)

sech unit has recognition helix, which increases specificity and stability

negative regulation

default for most bacterial is on, can be with inducible or repressible

repressor = protein blocks RNA poly from binding / moving along

operators = binding sites on DNA bind repressors near promoter

effectors = small molecule binds repressor and conformational change

positive regulation

genes with weak promoters may need activators

activators = enhance RNA poly-promoter interaction

activator binding sites, effectors

lac operon

3 genes do lactose metabolism,

in presence of lactose

• lactose > galactose + glucose

• lactose > allolactose

absence of glucose, adenylate cyclase makes cAMP

lac repressor

homotetramer, dimer of dimers

no lactose, repressor binds O1 and O2/O3

lactose > allolactose binds repressor to prevent it binding to DNA

CRP activator

lac operon has weak promoter

CRP

cAMP receptor protein;

contains binding sites for DNA and cAMP

homodimer, binds activator-binding site upstream promoter, interacts with RNA poly alpha

regulation of lac operon

negative =

• lacI gene upstream of operon, encodes repressor

• lac repressor binds operon in absence of allolactose

• operator site at transcription start site

positive =

• cAMP receptor protein binds DNA in presence of inducer cAMP

• activator binding site located upstream of the lac promoter

lac operon

visual

replication v transcription primers

RNA polymerases in transcription don’t use primers

who binds to the operator

repressors, NOT effectors (allolactose, which binds to proteins) or activators (which bind to enhancers or bind sites)