Transcription, RNA Processing, and

Transcriptional RegulationA. Structure of RNAB. Major Classes of RNAC. Transcription in ProkaryotesD. Transcription in EukaryotesE. Post-transcriptional Processing of Eukaryotic mRNAF. Transcriptional Regulation in Prokaryotes:

the Lac Operon as an exampleG. Transcriptional Regulation in Eukaryotes:

Steroid Hormones as an Example

A. Structure of RNA1. Uracil instead of Thymine2. Ribose instead of Deoxyribose3. Usually single-stranded4. May have hairpin loops (e.g. loops in

tRNA)

B. Major Classes of RNA1. Messenger RNA

mRNA Contains information for the amino acid

sequences of proteins2. Transfer RNA

tRNA Attaches to an amino acid molecule and

interfaces with mRNA during translation3. Ribosomal RNA

rRNA Structural component of ribosomes

B. Major Classes of RNA4. Small nuclear RNA

snRNA Component of small ribonucleoprotein

particles Processing of mRNA

5. Small nucleolar RNA snoRNA Processing of rRNA

6. Small cytoplasmic RNAs Variable functions; many are unknown

B. Major Classes of RNA7. Micro RNA

miRNA Inhibits translation of mRNA

8. Small interfering RNA siRNA Triggers degradation of other RNA molecules

9. Piwi-interacting RNA piRNA Thought to regulate gametogenesis

C. Transcription in Prokaryotes1. 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”2. 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 direction

C. Transcription in Prokaryotes

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

4. Nascent RNA strand synthesis (elongation) occurs only in the 5’ 3’ direction, with new nucleotides added to the 3’ end of the nascent strand

5. Transcription is catalyzed by DNA-directed RNA polymerases

C. Transcription in Prokaryotes

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

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

8. 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 pairs

C. Transcription in Prokaryotes

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

10. Bacterial RNA polymerase core has 4 or 5 subunits

α2ββ‘ω α2ββ‘ is essential; ω is not

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

C. Transcription in Prokaryotes

11. … for example: In E. coli, during log (exponential) growth, the

major sigma factor present is σ70

During stationary phase, it is σS

Shifting from σ70 to σS activates the transcription of multiple genes linked to survival during stationary phase

12. Transcription begins when the core RNA polymerase attaches to a sigma factor to form a holoenzyme molecule

C. Transcription in Prokaryotes

13. The holoenzyme binds to a promoter, and the dsDNA template begins to unwind

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

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

16. RNA polymerase moves 3’ 5’ along the template, synthesizing the nascent RNA 5’ 3’

C. Transcription in Prokaryotes

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

18. Some terminators require a termination factor protein called the rho factor (); these are rho-dependent. Others are rho-independent.

19. 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 monocistronic

D. Transcription in Eukaryotes

1. Chromatin in eukaryotes is unfolded to permit access to the template DNA during transcription

2. 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 regionUsually contains:TATA box – Consensus sequence at -25 to -30and other core consensus sequences

D. Transcription in Eukaryotes

2. … Regulatory promoter region

Immediately upstream from the core promoter, from about -40 to -150Consensus sequences include:OCT boxGC boxCAAT box

D. Transcription in Eukaryotes

3. 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 termination

E. Post-Transcriptional Processing of Eukaryotic mRNA1. 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.

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

E. Post-Transcriptional Processing of Eukaryotic mRNA3. Introns are removed, and the exons are

spliced together, by ribonucleoprotein particles called spliceosomes.

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

5. 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 linkage

E. Post-Transcriptional Processing of Eukaryotic mRNA6. The coding region ends with one or more

translational termination codons (stop codons).

7. At the 3’ end is a noncoding trailer region.

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

F. Transcriptional Regulation in Prokaryotes:

the Lac Operon as an Example1. Operon: A group of genes in bacteria

that are transcribed and regulated from a single promoter

2. 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 conditions

F. Transcriptional Regulation in Prokaryotes:

the Lac Operon as an Example3. 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)

F. Transcriptional Regulation in Prokaryotes:

the Lac Operon as an Example4. 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 promoter

F. Transcriptional Regulation in Prokaryotes:

the Lac Operon as an Example5. 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 transcription

F. Transcriptional Regulation in Prokaryotes:

the Lac Operon as an Example5. …

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

6. So … E. coli will metabolize glucose first, then lactose when the glucose runs out

G. Transcriptional Regulation in Eukaryotes:

Steroid Hormone as an Example1. Steroid hormones are secreted by endocrine

gland cells and travel through the bloodstream2. The steroid enters the cytoplasm of target cells

and binds to a cytoplasmic steroid receptor protein

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

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

G. Transcriptional Regulation in Eukaryotes:

Steroid Hormone as an Example5. 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.

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