Class Euk Trans Final

Eukaryotic Transcription

Types of RNAs Produced in Cells Types of RNAs

Functions

mRNAs

messenger RNAs, code for proteins

rRNAs

comprise ribosomes

tRNAs

adaptors between mRNA and amino acids in protein synthesis

snRNAs

splicing of pre-mRNAs

snoRNAs

process and chemically modify rRNAs

MicroRNAs

translation and mRNA degradation

Other non-coding RNAs

telomere synthesis, X-chromo. inactivation, protein transport

Four RNA Polymerases of Eukaryotic Cells Type of Polymerase

Genes Transcribed

RNA pol I

5.8S, 18S, and 28S rRNA genes

RNA pol II

protein coding genes, snoRNA genes, some snRNA genes, microRNAs

RNA pol III

tRNA genes, 5S rRNA genes some snRNA genes, genes for other small RNAs

RNA pol IV

plants only; small interfering RNAs (siRNAs)

Three types of RNA polymerase in eukaryotic nuclei Effect of α -amanitin

Type

Location

RNA synthesized

I II III

Nucleolus Nucleoplasm Nucleoplasm

Pre-rRNA for 18, 5.8 and 28S rRNAs Insensitive Pre-mRNA, some snRNAs Sensitive to 1 µ g/ml Pre-tRNAs, 5S rRNA, some snRNAs Sensitive to 10 µ g/ml

• α -amanitin from Amanita Phalloides binds tightly to RNA Pol II and blocks transcriptional elongation. • RNA Pol I transcribe 1 gene at ~200 copies. The gene for the 45S pre-rRNA is present in tandem array. • RNA Pol II transcribe ~25,000 genes; • RNA Pol III transcribe 30-50 genes at variable copy numbers.

(Also- Organelle RNAPs in Mitochondria and Chloroplasts. Encoded by organelle genomes. Similar to bacterial RNAPs.)

RNA Polymerase II •

RNA polymerase II (also called RNAP II or Pol II) transcribes DNA to synthesize precursors of mRNAs and most snRNAs.

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A 550 kDa complex of 12 subunits. A wide range of transcription factors are required for it to bind to its promoters and begin transcription.

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The largest subunit of Pol II (Rpb1) has a domain at its C-terminus that is called the CTD. Phosphorylation of the CTD is an important regulation mechanism, as this allows the binding and release of many factors that influence not only the transcription process, but also mRNA maturation and export from the nucleus. In this way, the CTD provides a platform for various factors that load on the nascent mRNA chain during transcription.

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The CTD consists of heptapeptide repeats (consensus: YSPTSPS) ranging from 26 in yeast and 52 in mammals, out of which serines and threonines get phosphorylated.

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Patterns of phosphorylation on these repeats can change rapidly during transcription. The regulation of the phosphorylation pattern and the resulting differential association of factors plays a major role not only in the regulation of transcription, but also in the fate of mRNA transcripts.

Subunit composition of  eukaryotic RNA polymerases •All three yeast polymerases have five  core subunits that exhibit some homology  with the β , β ‘, α  and ω  subunits in E.  coli RNA polymerase.  •RNA polymerases I and III contain the  same two non­identical α ­like subunits,  whereas polymerase II has two copies of a  different α ­like subunit. •All three polymerases share four other  common subunits. In addition, each RNA  polymerase contains three to seven unique  smaller subunits. •The largest subunit (1) of RNA  polymerase II also contains an essential C­ terminal domain (CTD). 27 (yeast) to 52  (human) copies of (YSPTSPS).  •Phosphorylation of CTD is important for  transcription and RNA processing.

Core Promoter Elements

Many genes, which are transcribed at low rates (e.g. genes encoding the enzymes required for basic metabolic processes required in all cells, often called “housekeeping genes”) do not contain a TATA box or an initiator. Most genes of this type contain a CG-rich region, or CpG island, of 20-50 nucleotides within ~100 base pairs upstream from the start site. Transcription of these genes can begin at any one of multiple possible sites over an extended region.

Basal (‘General’) Transcription Factors for RNA Polymerase II

Total: 43-44 polypeptides and over 2 million daltons.

TBP (TATA-box binding protein) •Conserved C-terminal domain of 180 amino acids. •A monomer with a saddle-shaped structure; the two halves show an overall dyad symmetry but are not identical. •Binds multiple transcription factors (TAFs, TFIIB and TFIIA). •Binds in the minor groove and significantly bends DNA.

Eukaryotic transcription cycle

Only the unphosphorylated RNA Pol II enters PIC.

The TFIIH complex has both helicase and kinase activities that can unwind DNA and phosphorylate the CTD tail of RNA Pol II. Release of TFIIE and then IIH during the synthesis of the initial 60-70nt.

Biochemical Reconstitution Revealed an Ordered Assembly of Factors for Initiation

Termination of Eukaryotic transcription • Type II genes: Transcription stops after AATAAA-Polyadenylation signal. • Type I genes:3-4 consecutive Ts • Type III genes: Stop after synthesis of serial Us.

Phosphorylation states of Pol II CTD during transcription cycle CTD

FCP1

Recycling

Pol II

Pol II

2 5 2 5 2 2 5 5

CTD

P-TEFb

TFIIH

CTD

Pol II

Pol II

5

CDK9

5 5 5

+1

PIC assembly

CycT1

CTD

Pol II 5’ cap

Promoter clearance & pausing for capping

2 5 2 5 2 2 5 5

Pol II

2 5 2 5 2 2 55

RNA

Release from pausing

Productive elongation

CTD heptapeptide repeats: 27­52 x (YS2PTS5PS)