Cell Biology Transcription, RNA, and Translation

RNA Polyamerase I

located in the nucleolus and synthesizes most rRNAs

RNA Polyamerase II

transcribes all protein coding genes (mRNA) and small nuclear RNAs (snRNAs) that function in splicing

RNA polyamerase III

functions outside the nucleolus, transcribes genes encoding tRNAs, rRNAs, snRNAs involved in splicing and protein transport

Promoters

determine initiation site and direct binding of RNA Pol

TATA Box

most common type of promoter sequence, 25-35 bp upstream of start site

Initiator Promoters

type of promoter sequence, C at -1 position and A at +1 position at start site

CpG islands

CG-rich stretch of 20-50 nucleotides about 100 bp from start, found in house keeping gases (Encode enzymes used in metabolism)

multi-subunit protein, TFIID binds to TATA sequence (TBP recognizes TATA)

Step 1 of transcription by Polymerase II

TFIIB binds then TFIIF/RNA Pol II binds

Step 2 of transcription by Polymerase II

Other TFs (TFIIE and H) bind forming transcription preinitiation complex

Step 3 of transcription by Polymerase II

DNA stands separate (TFIIH helices activity)

Step 4 of transcription by Polymerase II

RNA Polyamerase II starts transcription

Step 5 of transcription by Polymerase II

Soon after starting TFIIH kinase subunit phosphorylates RNA Pol II which causes all the TF except TFIID to disengage pol II

Step 6 of transcription by Polymerase II

TFIID

binds to TATA sequence in Pol II transcription

Transcription pre initiation complex

formed when TFs bind during Pol II transcription

TFIIH Kinase

phosphorylates RNA Pol II causing all TFs to disengage

TFIID

the only TF that doesn’t disengage Pol II in transcription

Phosphorylate

When TFIIH Kinase adds a phosphate group to RNA Pol II

Promoter-proximal elements

DNA sequence that controls transcription, has binding sites for transcription factors, lies 100-200 bp upstream of start site, may be cell type specific

Enhancer Sites

DNA sequence that controls transcription, 1000+ bp away from promoter, can be upstream of downstream of promoter or within an intron, has multiple transcription factor binding sites, bound TFs interact with Pol complex by looping connecting DNA out.

Activators

bind promoter-proximal elements and enhancer sites

DNA Binding Domain

Activators contain a single … (zinc finger motif) linked to one or more activation domains

Activation Domain

contacts transcription machinery and increases rate of initiation

Repressors

bind promoter-proximal elements only

Repressor domains

repressors have DNA binding domain and

Competition

way the rate of transcription is decreased, … between activator and repressor for same regulatory DNA sequence.

inhibits activation domain

way the rate of transcription is decreased, when both activator and repressor bind DNA but repressor … of activator protein

Transcription initiation complex

way the rate of transcription is decreased, repressors preventing … to form

Acetylation

… of N-terminal tails of histones loosens its association with DNA

Deacetylation

strengthens the histone/DNA association

5’capping

after RNA produced by pol II reaches 25-30 nucleotides, 7 methlyguanosine is added to the 5’ end, catalyzed by capping enzyme that associates with carboxyl term. tail of Pol II.

Cleavage Polyadenylation

coupled to termination of transcription

CPSF binds AU-ruch signal (AAUAAA), 10-30 nucleotides upstream Poly(A) site

Step 1 in Cleavage Polyadenylation

Other cleavage factors bind GU-Rich site in downstream signal

Step 2 in cleavage polyadenylation

Poly (A) polymerase (PAP) binds complex, cleavage of RNA at poly A site

Step 3 in Cleavage Polyadenylation

Cleavage factors and RNA fragment released then PAP adds 12 “A”s slowly

Step 4 in Cleavage Polyadenylation

Poly (A)-binding protein (PABPII) binds to short poly (A) tail accelerates PAP

Step 5 in Cleavage Polyadenylation

After 200-250 “A”s have been added PABPII signals to PAP to stop

Step 6 in Cleavage Polyadenylation

RNA Splicing

removal of introns from transcript

5’G of intron binds to A near 3’ end of intron (5’, 2’ phosphodiester bond)

Step 1 of RNA Splicing

lariat structure removed and 2 exons joined by phosphodiester bond

Step 2 of RNA Splicing

Catalyzed by spliceosome (5 snRNPs) base pairing with transcript

Step 3 of RNA Splicing

Entire reaction thought to involve 70 proteins and is very complex

Step 4 of RNA Splicing

Cap-binding protein

“export ready mRNA” are bound to

Poly-A binding protein

“export ready mRNA” are bound to

Exon junction complex (EJC)

“export ready mRNA” are bound to

Nuclear Pore Complex

recognizes and exports only completed and mature mRNAs

Translation

Process consists of translating every 2 nucleotides in mRNA into a corresponding AA, catalyzed by ribosomes in the cytoplasm

Ribosomes

large complexes of proteins and RNA, forms 2 subunits (small and large), exists free in cytoplasm and RER

Codon

3 nucleotides

tRNAs

molecule involved in translation, L-shaped, one end of L contains anticodon that binds to codon in mRNA and other end binds to specific amino acid

Aminoacyl-tRNA syntheses

molecule involved in translation, covalently links AAs to tRNAs

Peptidyl transferases

molecule involved in translation, catalyzes peptide bonds of incoming “AA”s (part of large ribosomal subunit)

initiator tRNA/Met binds to P site in small ribosomal subunit then scans mRNA for start codon (AUG) and binds there (complementary base pairing.

Step 1 of translation

large ribosomal subunit binds start site surrounding mRNA

Step 2 of translation

tRNA carrying next AA binds to A site in ribosome (next to P site) and peptide transferase catalyzes peptide bond between the 2 AAs

Step 3 of translation

initiator tRNA leaves P site and the tRNA that was in A site is transferred to the P site when ribosome moves 3 nucleotides down the mRNA

Step 4 of translation

Steps 3 and 4 are repeated until ribosome reaches a stop codon in the mRNA (UAA, UAG, UGA) then 2 release factors sit in A site, help cleave peptide chain from tRNA and ribosome subunits disengage

Step 5 of translation