Transcription and Translation of Prokaryotes

Central Dogma

DNA encodes genes which make up genomesI

In prokaryotes transcription and translation occur

simultaneously in the cytoplasm

Promoter

where promoter binds, determines where transcription starts, -35 to -10 region

Coding sequence

what encodes the proteinO

Open Reading frame

Coding sequence that contains a start but no stop codon

Regulatory sequences

tells polymerase when, where and how much

Transcription Terminator

Stops stranscription

\+1 Site

Where the first nucleotide is transcribed

Operon

A group of genes coding for the same function

Each gene in the operon has

its own start and stop codon

Introns

get cut out of the sequence

Extrons

are kept

5 PPP replaces

need for 3’ OH

5 PPP binding

one of the P binds to the O in 3' spot, releasing PPi and creating a bond with the next nucleotide withou t primer

RNA synthesis goes from

5’ to 3’, adding to 3’

Template strand

Strand that is used for transcription

Coding strand

Strand not used for transcription

RNA sequence is almos identical to

coding strand

RNA Polymerase goes from

5’ to 3’, adding to 3'

mRNA messengers

encode proteins

tRNA proteins

transfers amino acids to mRNA

rRNA

ribosomal RNA

sRNA

small RNA that regulates gene expression

functional/structual RNAs

tRNA and rRNA

Template strand always goes from

3’ to 5’

Stages of Transcription in Bacteria

Initiation, Elongation, Termination

Initiation

template strand is prepared for transcription

Elongation

rNTPs are added on by RNA polymerase

Termination

transcription finishes and RNA polymerase disassociates

Transcription step 1

RNA polymerase binds to the promoter region

transcription step 2

DNA helix is unwond to form a transcription bubble that starts RNA polymerase

transcription step 3

one of the strands is used as the template strand--bases are added from 5’ to 3’

transcription step 4

transcription ends due to either intrinsic mechanisms or rho dependent mechanisms

RNA polymerase alpha subunit

enzyme assembly, promotes interactions with regulatory proteins

RNA polymerase beta subunit

catalysis

RNA polymerase sigma subunit

recognizes promoter sequence and binds to the region

RNA polymerase location in alpha helixes

major grooves

RNA polymerase sigma dissociates

after elongation begins

Holoenzyme

functional protein that initiates transcription

Higher promoter strength

higher degree that promoter sequence matches consensus sequence

Consensus sequence

TATAAT at -10 and TTGACAT at --35

RNA polymerase has a ___ fidelity that DNA polymerase

lower

2 types of transcription termination

intrinsic mechanism and rho-dependent mechanism

Intrinsic mechanism definition

stops transcription without need of proteins, DNA encodes the stop alone

intrinsic mechanism process

region of G and C are encoded and then form strong noncovalent bonds with each other, which causes a hairpin loop to form. After passing a region of A, it encodes for U which is weak, causing the the strong G and C bonds to pop RNA polymerase off of the DNA strand

hairpin loop is formed by

Strong G-C bonds forming and then a region of weak U nucleotides

Rho dependent mechanism definition

termination that requires the presence of rho-protein to end transcription

Rho depent mechanism process

Rho binds to the the RUT on the RNA and races the paused RNA polymerase. Once it catches up at the transcription bubble, it pulls the RNA and sends RNA polymerase flying off

Rho dependent mechanism causes

hydrolyzation of ATP into ADP and Pi

In transcription termination signals are

in 3’ untranslated region

basal expression

RNA polymerase transcription only, low expression

Negative regulation

repressor binds to operator and blocks RNA polymerase, no expression

Inducers

Allosteric effectors that bind to the regulatory proteins

Induces can cause repressors to

not bind

Inducers can cause activation of acitvator and

rejection of repressor

Positive regulation

activator is present with RNA polymerase, high expression

In positive regulation, inducers

bind to repressors, preventing them from binding to operator

lac I

codes for repressors

lac Z

beta-galactosidase, turns lactose into glucose and galactose

lac Y

permease, allows lactose into the cell

lac A

transacetylase, breaks down sugars

IPTG

inducer that doesn’t need permease

Negative regulation occurs in

the absence of lactose

types of mutants

structural genes and regulatory mutants

Structural gene mutants

change the mRNA expression, ie. lacZ- or lacY-

Regulatory mutants

Mutant in promoters, operators, or repressor producers. ie lacI-

Partial diploid mutants

have a second compy of the lac operon on a plasmid, restoration of some functions

cis defintion

location matters, transcription factor needs to be on the strand and close to the thing its transcribing

cis examples

promoters, operators, lacZ, lacY, lacA

trans

location doesn’t matter and transcription factor can be anywhere

trans examples

lacI, activator

Cyclic amp regulation

the presence of glucose determines whether or not activator will bind or not

Glucose exists

transcription of lactose enzymes is low

glucose is a better

energy source that lactose because it doesn’t take as much energy to catalyze

Glucose not present

transcription occurs at a high rate because CAP is activated and CAMP is high

CAP activation

RNA polymerase binds more often

helix-turn-helix DNA binding domain

NHEJ- Non homologous end joining

imprecise insertions or deletions causing loss of function

Homology directed repair

precise changes in nucleotide sequence that uses donor sequence

homologous recombination requires

high homolog

Redundancy

multiple tRNA molecules with different anticodons carry the same amino acid

Wobble Base Pairing

Single tRNA reads 2 different codes for the same amino acid

Inosine pairs with

A, U, C

Inosine is found in

anticodon loop of some tRNA moleculesI

Inosine is

deamination product of A made after tRNA transcription

ribosome in translation

binds mRNA and tRNA-AA. Reads instructions for protein synthesis from mRNA

3 binding site

Acceptor, Peptidyl, and Exit

Acceptor

where incoming amino acids are accepted

Peptidyl

where tRNA’s amino acid bonds with the growing peptide chain

Exit

Empty tRNAs are sent out of the mRNA

Amino acid chain is polymerized from N terminus to C terminus

Amino acids are added to COO-

tRNA function translation

reads genetic code and brings amino acid to the codon

Anti-codon 3’ to 5’ interacts with

codon’s 5’ to 3’

Amino acyl tRNA-synthases

Enzymes that link specific amino acids to the tRNA that read the codons. Connects amino acid’s carboxyl group to tRNA’s 3’ OH group

Aminoacyl tRNA

charged RNA

Initiation Factor 3

separates 50s and 30s

IF2

delivers initiator tRNA charged with fMet to the P site, establishing the open reading frame