L16 Transcription and RNA Processing

How is RNA polymerase similar to DNA polymerase?

-Requires ssDNA template (DNA must be unwound)

-Requires the four NTPs

-Same primer extension reaction (produces PPi)

How is RNA polymerase different from DNA polymerase?

-No primer is required

-No sliding clamp

-Ribo-NTPs not dNTPs (and UTP instead of dTTP)

-No proofreading activity

Also:

-Transcription, after initiation, only goes in one direction (no bi-directional origin) and on one strand (no fork)

-Transcription can occur on either strand

-Transcription must be terminated by specific sequences

What is the function of Ribosomal RNA (rRNA)?

Translation (ribosome structure and catalytic activity)

What is the function of Transfer RNA (tRNA)?

Delivery of amino acids to ribosomes during translation

What is the function of small interfering RNA (siRNA)?

Sequence-specific inactivation of mRNA

What is the function of Micro RNA (miRNA)?

Sequence-specific inactivation of mRNA

What is the function of Large intergenic noncoding RNA (lineRNA)?

Transcriptional control

What is the function of Small nucleolar RNA (snRNA)?

RNA splicing

What is the function of Small nucleolar RNA (snoRNA)?

Sequence-specific methylation of rRNA

Coding DNA strand

The "coding" DNA strand sequence and polarity are the sam as RNA except T in DNA for U in RNA

"Non-coding" DNA strand

The "non-coding" DNA strand is involved only as template in the process of transcription

When both DNA strands of a gene are shown, which is typically the coding gene?

The "coding" DNA strand is typically written above non-coding

When only one strand of transcribe DNA is show which one is it?

The "coding" strand (unless explicitly indicated otherwise)

Genes in E. coli are typically contained within what?

operons

-transcription is coregulated

-single transcript to yiel dpolycistronic RNA encoding more than one polypeptide

What are the three stages of transcription?

initiation, elongation, termination

What is initiation?

Initiation involves the binding of RNA polymerase to the promoter

What is the promoter?

(regulatory sequence upstream of a gene)

What is the Consensus sequence?

Average of a number of closely related but nonidentical sequences

What is the significance of the sequences at -10 (Prinbow box) and -35?

They are important in prokaryotic transcription, specifically initiation

What does RNA Polymerase (RNAP) holoenzyme do?

RNA Polymerase holoenzyme slides down the DNA until it reaches a promoter sequence

How do rates of transcription vary?

They vary with rates of complex formation with RNA polymerase holoenzyme

What is the sigma factor critical for?

The sigma or specificity factor is critical for binding RNAP holoenzyme at promoter

Is the enzyme-promoter complex considered opened or closed at the start of transcription?

Open

What signals the end of the initiation phase?

After synthesis of ~10 nucleotides, sigma factor is released reducing affinity for promoter

What happens in the elongation phase?

In elongation phase, active transcription complex moves "downstream" of promoter

What is the transcription bubble?

12-14 bp of unwound DNA with DNA:RNA hybrid

(+) supercoils ahead

(-) supercoils behind

-~30 bp total in RNAP at a time

What happens during Elongation?

Multiple mRNA can be transcribed concurrently

What are the two types of transcription termination in bacteria?

Intrinsic Termination and Rho-dependent termination

What is Intrinsic termination?

RNA synthesis is terminated after transcription of an inverted repeat sequence

-Stable hairpin forms that causes RNA polymerase to slow or stop

-TNA is released due to weak base-pair interactions

What is Rho-depemdent termination?

RNA synthesis is terminated with aid of ATP-dependent helicase rho factor

When does mRNA translation begin?

As soon as ribosome binding site is exposed

iClicker Question:

One difference between transcription and DNA replication is

Only one strand is transcribed while both strands are replicated

What does RNA pol I do in eukaryotes?

transcribes rRNA genes in nucleoli

What does RNA pol II do in eukaryotes?

synthesizes mRNA precurors

What does RNA pol III do in eukaryotes?

synthesizes tRNA, 5S rRNA, and snRNAs

How many RNA polymerases does the mitochondria use for gene transcription.

Mitochondria and prokaryotes both use a single RNA polymerase for all gene transcription

What doe eukaryotic RNA polymerases require at promoters to initiate transcription?

transcription factors

RNA Polymerase II

-Has a greater subunit complexity than prokaryotic RNA polymerase homolog

-claw-like shape

-2 Mg2+ ions in the active site

What is the Rpb1 subunit?

It contains the active site

-also contains c-terminal domain (CTD)

-Initiates when unphosphorylated and moved to elongation only when phosphorylated

-Contains "bridge" helix

created Brownian ratchet to move enzyme forward

What is the Rpb2 subunit?

Functions as a clamp

-promotes precessivity

-portion creates a 'wall' that directs template strand out at a 90 degree turn

-recognizes both proper base pairing and the ribose through extensive H-bonding

-strogly discriminates against dNTPs

Does eukaryotic RNAP have a removable sigma factor?

No

What are core promoter elements (CPEs)

They allow binding of the transcription machinery

-TATA box is a well studied CPE

-35-50 bases tatal generally located ~30 to +6 relative to the start site

Many genes have what between -80 and -90?

conserved CCAAT (CCAAT box) that influences transcription rate

What do promoters lacking TATA box use?

MTE-motif ten element or DPE element

What is TBP?

TATA box binding protein

-binds the TATA box as the first step in initiation

-subunit of TFIID transcription factor

What does THIID and THIIB binding at core promotor do?

recruits additional factors and RNA polymerase II

What are the general transcription factors required for all protein encoding mRNAs?

-6 protein/protein complexes: TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH

-minimal number of additional proteins required for transcription

What is TFIIH?

An ATP-dependent helicase

Explain the formation of Pre-initiation complex (PIC)

1.The TBP component of TFIIS binds to the TATA box of the promoter

2.TFIIA and TFIIB bind

3.TFIIF binds to RNAP I and escorts it to the complex

4.TFIE and TFIH are sequentially recruited, thereby completing the PIC

Are the initiral RNAs produced by transcription (primary transcripts) functional?

Not necessarily

-many are altered by posttranscriptional modification

What is postranscriptional modification?

-Exo and ndo nuclease modified to remove bases

-Addition of bases to both 5' and 3' ends

-Modification of specific nucleotide residies

mRNA modification in prokaryotes vs eukaryotes

Prokaryotes:

most mRNAs are translated w/o further modification

-translation can occur co-transcriptionally

Eukaryotes

mRNA modification is extensive in eukaryotes

-synthesized in the nucleus yet translation occurs in the cytosol

Eukaryotic mRNAs have a unique modification added to the 5' end

7-methyguanosine Cap

-After transcript is ~30 bases ling

-phosphate removal by RNA triphosphate

-guanylylation by transferase

-they methylation

-cap is bound to the cap binding complex (CBC)

-Protects 5' end from exonucleases

-Associates with the phospho-CTD to switch RNA pol II from initiation to elongation

In transcription termination in eukaryotes precise?

No it is imprecise

Mature eukaryotic mRNAs have 3' ends of poly(A) tails of ow many bases?

~250 bases (~80 t in yeast)

-occurs co-transcriptionally

What is the Poly(A) tail synthesized by?

poly(A) polymerase (PAP)

-activated by cleavage and polyadenylation specificity factor (CPSF)

-PAP synthesizes poly(A) tail from ATP w/o a template

What is Poly (A) length associated with

cytosolic lifetime

-shortens with age

-binds poly(A)-binding protein (PABP) which protects against nucleases