Transcription, RNA Processing, and Transcriptional Regulation - PowerPoint Presentation

Slide1

Transcription, RNA Processing, and Transcriptional Regulation

Structure of RNA

Major Classes of RNA

Transcription in Prokaryotes

Transcription in Eukaryotes

Post-transcriptional Processing of Eukaryotic mRNA

Transcriptional Regulation in Prokaryotes:

the Lac Operon as an example

Transcriptional Regulation in Eukaryotes:

Steroid Hormones as an ExampleSlide2

A. Structure of RNAUracil instead of Thymine

Ribose instead of Deoxyribose

Usually single-stranded

May have hairpin loops (e.g. loops in tRNA)Slide3

B. Major Classes of RNA

Messenger RNA

mRNA

Contains information for the amino acid sequences of proteins

Transfer RNA

tRNA

Attaches to an amino acid molecule and interfaces with mRNA during translation

Ribosomal RNA

rRNA

Structural component of ribosomesSlide4

B. Major Classes of RNASmall nuclear RNA

snRNA

Component of small ribonucleoprotein particles

Processing of mRNA

Small

nucleolar

RNA

snoRNA

Processing of rRNA

Small cytoplasmic RNAs

Variable functions; many are unknownSlide5

B. Major Classes of RNAMicro RNA

miRNA

Inhibits translation of mRNA

Small interfering RNA

siRNA

Triggers degradation of other RNA molecules

Piwi

-interacting RNA

piRNA

Thought to regulate gametogenesisSlide6

C. Transcription in Prokaryotes

Requires a double-stranded DNA template

The DNA strands separate, and only one of the strands is used as a template for transcription

“Template strand” and “nontemplate strand”

Direction and numbering conventions

From

the 3’

 5’ direction on the template strand is called “downstream”

From the 5’

 3’ direction on the template strand is called “upstream”

The nucleotide at the transcriptional start site is designated “+1” and the numbering continues +2, +3, etc. in the downstream direction

The nucleotide immediately upstream from +1 is designated “-1” (there is no 0); numbering continues -1, -2, etc. in the upstream directionSlide7

C. Transcription in Prokaryotes

Transcription requires nucleoside triphosphates (NTPs; ATP, GTP, CTP, UTP) as raw materials

Nascent RNA strand synthesis (elongation) occurs only in the 5’

 3’ direction, with new nucleotides added to the 3’ end of the nascent strand

Transcription is catalyzed by DNA-directed RNA polymerasesSlide8

C. Transcription in Prokaryotes

The initiation of transcription occurs when RNA polymerase binds to a “promoter region” upstream from the transcriptional start site

Promoter regions typically have short stretches of common nucleotide sequences, found in most promoters, called “consensus sequences”

Common prokaryotic (bacterial) consensus sequences include:

-10 consensus sequence: TATAAT box or

Pribnow

box

-35 consensus sequence: TTGACA

-40 to -60: Upstream element; repetitive A-T pairsSlide9

C. Transcription in Prokaryotes

Bacterial RNA polymerase consists of a core enzyme and a sigma factor

Bacterial RNA polymerase core has 4 or 5 subunits

α

2

ββ

‘

ω

α

2

ββ

‘ is essential;

ω

is not

Sigma factors (

σ

) are global regulatory units. Most bacteria possess several different sigma factors, each of which mediate transcription from several hundred genes …Slide10

C. Transcription in Prokaryotes

… for example:

In

E. coli

, during log (exponential) growth, the major sigma factor present is

σ

70

During stationary phase, it is

σ

S

Shifting from

σ

70

to

σ

S

activates the transcription of multiple genes linked to survival during stationary phase

Transcription begins when the core RNA polymerase attaches to a sigma factor to form a holoenzyme moleculeSlide11

C. Transcription in Prokaryotes

The holoenzyme binds to a promoter, and the

dsDNA

template begins to unwind

A nascent RNA strand is started at +1 on the template

After transcription is initiated, the sigma factor often dissociates from the holoenzyme

RNA polymerase moves 3’

 5’ along the template, synthesizing the nascent RNA

5’  3’Slide12

C. Transcription in Prokaryotes

Transcription ends (termination) when RNA polymerase reaches a terminator sequence, usually located several bases upstream from where transcription actually stops

Some terminators require a termination factor protein called the rho factor (

); these are rho-dependent. Others are rho-independent.

Messenger RNA in bacteria is often polycistronic, which means that it has the code for >1 protein on a single mRNA molecule; mRNA in eukaryotes is almost always monocistronicSlide13

D. Transcription in Eukaryotes

Chromatin in eukaryotes is

u

nfolded to permit access to the template DNA during transcription

Eukaryotic promoters

Recognized by accessory proteins that recruit different RNA polymerases

(I, II, or III)

Consist of a core promoter region and a regulatory promoter region

Core promoter region is immediately upstream from the coding region

Usually contains:

TATA box – Consensus sequence at -25 to -30

and other core consensus sequencesSlide14

D. Transcription in Eukaryotes

…

Regulatory promoter region

Immediately upstream from the core promoter, from about -40 to -150

Consensus sequences include:

OCT box

GC box

CAAT boxSlide15

D. Transcription in Eukaryotes

Eukaryotic RNA polymerases

RNA polymerase I: Synthesizes pre-

rRNA

RNA polymerase II: Synthesizes pre-mRNA

RNA polymerase III: Synthesizes tRNA, 5S rRNA, and several small nuclear and cytosol RNAs

Also, the different RNA polymerases use different mechanisms for terminationSlide16

E. Post-Transcriptional Processing of Eukaryotic mRNA

In eukaryotes, mRNA is initially transcribed as precursor mRNA (“pre-mRNA”). This is part of a transcript called heterogeneous nuclear RNA (

hnRNA

); the terms

hnRNA

and pre-mRNA are sometimes used

interchangably

.

Almost all eukaryotic genes contain introns: noncoding regions that must be removed from the pre-mRNA. The coding regions are called exons.Slide17

E. Post-Transcriptional Processing of Eukaryotic mRNA

Introns are removed, and the exons are spliced together, by ribonucleoprotein particles called spliceosomes.

mRNA contains a “leader sequence” at its 5’ end, before the coding region. The coding region begins with a translational initiation codon (AUG).

A methylated guanosine cap is added to the 5’ end of the mRNA by capping enzymes. The cap is attached by a 5’

 5’ triphosphate linkageSlide18

E. Post-Transcriptional Processing of Eukaryotic mRNA

The coding region ends with one or more translational termination codons (stop codons).

At the 3’ end is a noncoding trailer region.

A 3’ poly-A tail, consisting of 50 – 250 adenosine nucleotides, is added to the 3’ end by a 3’ terminal transferase enzyme.Slide19

F. Transcriptional Regulation in Prokaryotes: the Lac Operon as an Example

Operon: A group of genes in bacteria that are transcribed and regulated from a single promoter

Constitutive vs. regulated gene expression

Constitutive gene expression: When a gene is always transcribed

Regulated gene expression: When a gene is only transcribed under certain conditionsSlide20

F. Transcriptional Regulation in Prokaryotes: the Lac Operon as an Example

The lac operon in

E. coli

consists of:

3 structural genes (genes that encode mRNA)

lac z

gene: Encodes

β

-

galactosidase

lac y

gene: Encodes

β

-

galactoside

permease

lac a

gene: Encodes

β

-

galactoside

transacetylase

The lac promoter gene:

lac p

The lac repressor gene:

lac i

(constitutively expressed and transcribed from its own promoter, different from

lac p

)

The lac operator region:

lac o

(which overlaps

lac p

and

lac z

)Slide21

F. Transcriptional Regulation in Prokaryotes: the Lac Operon as an Example

The genes of the lac operon are only transcribed in the presence of lactose (or another chemically similar inducer)

In the absence of lactose, the lac repressor protein binds to

lac o

(lac operator) and blocks RNA polymerase from binding to the promoter (

lac p

)

In the presence of lactose:

Lactose in the cell is converted to

allolactose

Allolactose

binds to the lac repressor protein, causing it to causing it to dissociate from the operator so RNA polymerase can reach the promoterSlide22

F. Transcriptional Regulation in Prokaryotes: the Lac Operon as an Example

Transcription of the lac operon is stimulated by conditions of low glucose concentration

When glucose levels are low:

Adenylate

cyclase

activity is high and the concentration of cyclic AMP (

cAMP

) is high

cAMP

binds to the

catabolite

activator protein (CAP)

The

cAMP

/CAP complex increases the efficiency of binding of RNA polymerase to the promoter

So there is increased lac transcriptionSlide23

F. Transcriptional Regulation in Prokaryotes: the Lac Operon as an Example

…

When glucose levels are high:

Adenylate

cyclase

activity is lowered, so

cAMP

levels are low

This means there is much less

cAMP

/CAP complex

And there is decreased lac transcription

So …

E. coli

will metabolize glucose first, then lactose when the glucose runs outSlide24

G. Transcriptional Regulation in Eukaryotes: Steroid Hormone as an Example

Steroid hormones are secreted by endocrine gland cells and travel through the bloodstream

The steroid enters the cytoplasm of target cells and binds to a cytoplasmic steroid receptor protein

The steroid receptor/steroid complex enters the nucleus, where it binds to regulatory sites (typically upstream from specific promoters)

Transcription from some promoters may be activated (“turned on”) while transcription from other promoters may be inhibited (“turned off”)Slide25

G. Transcriptional Regulation in Eukaryotes: Steroid Hormone as an Example

Once the genes that have been activated by the steroid receptor/steroid complex (primary response or early genes) have been transcribed and translated, some of the proteins may act to regulate the expression of other genes (secondary response genes), etc.

So … you may have a series of different transcriptional events over a time course with early, middle, and late events