DNA Technology Lect 8

8/12/2019 DNA Technology Lect 8

1/21

The basic principle of regulation in bacteria is that gene expression is

controlled by a regulator that interacts with a specific sequence orstructure in DNA or mRNA at some stage prior to the synthesis of

protein.

The stage of expression that is controlled can be transcription, when

the target for regulation is DNA, or it can be at translation, when thetarget for regulation is RNA.

When control is during transcription, it can be at initiation or at

termination. The regulator can be a protein or an RNA.

"Controlled" can mean that the regulator turns off (represses) the

target or that it turns on (activates) the target. Expression of many

genes can be coordinately controlled by a single regulator gene on the

principle that each target contains a copy of the sequence or structure

that the regulator recognizes.

Regulatory RNA


2/21

Regulators may themselves be regulated, most typically in response to

small molecules whose supply responds to environmental conditions.

Regulators may be controlled by other regulators to make complex

circuits.

Regulation via RNA uses changes in secondary structure as the

guiding principle. The changes in structure may result from either

intramolecular or intermolecular interactions.

The most common role for intramolecular changes is for an RNA

molecule to assume alternative secondary structures by utilizing

different schemes for base pairing. The properties ofthe alternative

conformations may be different.

Secondary structure also is used to regulate the termination of

transcription, when the alternative structures differ in whether they

permit termination.


3/21

A regulator RNA is a small RNA with a single-stranded region th at can pair with

a sing le-stranded regio n in a target RNA.

In intermolecular interactions, an RNA regulator recognizes its target by

the familiar principle of complementary base pairing


4/21

The trp operon consists of five structural genes arranged in a contiguous

series, which code for the three enzymes (anthranilate synthetase, indol-glycerol synthetase, and tryptophan synthetase) that convert chorismic

acid to tryptophan.

The operon starts at a promoter at the left end of the cluster, trp operon

expression is controlled by two separate mechanisms:

1- Repression of expression is exercised by a represser protein (coded

by the unlinked gene trpR) that binds to an operator that is adjacent to the

promoter.

2- Attenuation controls the progress of RNA polymerase into the operon

by regulating whether termination occurs at a site preceding the first

structural gene.

The E.colitryptophan operon


5/21

The trp operon c onsists of f ive cont iguou s s tructura l genes preceded

by a control region that includes a promoter, operator, leader peptide

cod ing region, and attenuator.


6/21

Attenuation was first revealed by the observation that deleting a

sequence between the operator and the trpE coding region can increase

the expression of the structural genes.

This effect is independent of repression: both the basal and

derepressed levels of transcription are increased. Thus this site

influences events that occur afterRNA polymerase has set out from the

promoter (irrespective of the conditions prevailing at initiation).

An attenuator (intrinsic terminator) is located between the promoter and

the trpE gene. It provides a barrier to transcription into the structural

genes.

Termination at the attenuator respond to the level of tryptophan. In the

presence of adequate amounts of tryptophan, termination is efficient. In

the absence of tryptophan, RNA polymerase can continue into the

structural genes.


7/21

An attenuator contro ls the

prog ression of RNAP into the

trp g enes. RNAP init iates at theprom oter and then proceeds.

In the absence of tryp toph an,the polymerase cont inue into

the stru ctural genes.

In the presence of tryp toph an,

there is ~ 90% pro babi l i ty of

Termination.

ribosome


8/21

Eukaryotic transcription is more complex than prokaryotic transcription

and, until recently, it has seemed that every eukaryotic gene was unique

requiring its own transcription machinery.

However, it is now possible to simplify the story somewhat. The

promoters for different genes are different. Each contains a combinationof sites to which specific protein factors bind. All of these factors help

RNA polymerase to bind in the correct place and to initiate transcription.

However, the repertoire of transcription factors and transcription factor

binding sites is not unlimited.

There are three distinct RNA polymerases in a eukaryotic cell nucleus

which define the three major classes of eukaryotic transcription unit:

Transc r ipt ion in eukaryot ic cells


9/21

Because there is no nucleus to separate the processes of transcription

and translation, when bacterial genes are transcribed, their transcripts

can immediately be translated.

Prokaryotic cells


10/21

Transcription and translation are spatially and temporally separated ineukaryotic cells; that is, transcription occurs in the nucleus to produce a

pre-mRNA molecule.

The pre-mRNA is typically processed to produce the mature mRNA, which

exits the nucleus and is translated in the cytoplasm.

Eukaryotic cells


11/21

mRNA in Eukaryotes

The sequence of a eukaryotic protein-coding gene is typically not

colinear with the translated mRNA; that is, the transcript of the gene is a

molecule that must be processed to remove extra sequences (introns)

before it is translated into the polypeptide.

Most eukaryotic protein-coding genes contain segments called introns,

which break up the amino acid coding sequence into segments calledexons.

The transcript of these genes is the pre-mRNA (precursor-mRNA).

The pre-mRNA is processed in the nucleus to remove the introns andsplice the exons together into a translatable mRNA. That mRNA exits the

nucleus and is translated in the cytoplasm.


12/21

Synthesis of m ature mRNA in eukaryot ic cells


13/21

Pre-mRNA Processing (Splicing)

The steps of pre-mRNA splicing (intron removal) are as follows:

The intron loops out as snRNPs (small nuclear ribonucleoprotein particles,

complexes of snRNAs and proteins) bind to form the spliceosome.

The intron is excised, and the exons are then spliced together.

The resulting mature mRNA may then exit the nucleus and be translated in the

cytoplasm.


14/21


15/21

The genomic DNA contains all the information for the structure and

function of an organism.In any cell, only some of the genes are expressed,

that is, transcribed into RNA.

There are 4 types of RNA, each encoded by its own type of gene:

mRNA - Messenger RNA: Encodes amino acid sequence of a polypeptide.

tRNA - Transfer RNA: Brings amino acids to ribosomes during translation.

rRNA - Ribosomal RNA: With ribosomal proteins, makes up the

ribosomes, the organelles that translate the mRNA.

snRNA - Small nuclear RNA: With proteins, forms complexes that areused in RNA processing in eukaryotes. (Not found in prokaryotes.)


16/21


17/21

Eukaryo t ic RNA po lymerases

polymerase location type of RNA transcribed

I nucleus/nucleolus rRNA (except for 5S rRNA)

II nucleus hnRNA (i.e. pre-mRNA)

III nucleussmall RNA such as tRNA and 5S

rRNA


18/21

In eukaryotes there are five different RNA polymerases.

RNA polymerase Ihas become specialized for transcription of the

genes for the large ribosomal RNAs.

Eukaryotic cells need massive amounts of ribosomal RNA and they have

many copies of ribosomal RNA genes.

RNA polymerase IIis responsible for transcribing protein-encoding

genes to produce mRNA.

It has evolved some special features that allow it to be coupled to the

processing of mRNA precursors. Unlike bacteria mRNA, eukaryotic mRNA

is modified at the 5 and 3 ends and the mRNA precursor can be spliced.


19/21

RNA polymerase II can transcribe RNA from nicked dsDNA templates

or from ssDNA templates. However, by itself, it cannot initiate

transcription at a promoter. In this respect, it resembles the core form of

bacterial RNA polymerase.

The eukaryotice enzymes also interact with a greater variety of

transcription factors. The RNAP II core enzyme is associating with

several transcription factors (TF) that are required for transcription

initiation.

RNA polymerase IIImakes transfer RNA (tRNA), small ribosoma RNA

(5S RNA) and most of the small RNAs that make up the fourth class of

RNA.

The 4th and 5th types of eukarytic RNA polymerases are the

mitochondrial and chloroplast versions.


20/21

RNA po lymerase I prom oter region

The RNA polymerases promoters


21/21

RNA polymerase III co re promoter

Documents

DNA Technology Lect 8