19. mRNA processing

in e.coli transcription termination - 2 mechanisms

1. Rho-independent: RNA polymerase only

2. Rho-dependent: RNA polymerase + protein RHO

Premature termination = attentuation

rho dependent termination

1. rho binds to rut site n mRNA

2. RNAP pauses at termination site → provides time for rho to catch up (hairpin required)

3. rho has no affinity for RNAP, tracks along mRNA until it catches up

4. rho unwinds DNA-RNA hybrid (requires ATP)

5. mRNA, rho, RNAP all released

RHO independent termination sites

1. GC rich self-complementary region with several intervening nucleotides, when transcribed - forms stem loop structure

2. followed by series of U resides

Rho independent termination

1. a rho independent terminator contains an inverted repeat followed by a string of approximately 6 adenine nucleotides

2. the inverted repeats are transcribe into RNA

3. inverted repeats in RNA fold into a hairpin loop, which causes RNA polymerase to pause

4. RNA transcript separates from the template, terminating transcription

DNA sequence where RNA polymerase either terminates or continues with transcription

not a termination site (t site)

attenuation in the trp operon

regulatory mechanism where transcription stops early if trp levels are high, and continues if trp levels are low

expression of trp operon controlled at two levels

1. trpR gene repressor, in the presence of tryptophan binds the operator (o)

2. attenuator sequence that prematurely terminates transcription when high levels of trp are present (attenuated RNA consists of only a short leader sequence)

RNAP I requires a

polymerase-specific termination factor that binds to a specific DNA sequence

RNAP III terminates after

transcribing a series of U resides but does not require a stem-loop structure

RNAP II terminates due to

3’ processing of the transcript - polyadenylation by protein complex that is carried by the phosphorylated CTD tail and binds to elements in 3’ UTR of mRNA

heterogenous nuclear RNA

transcripts made by RNAPII before they are fully processed

RNP motif - RBD (Rna Binding Domain) most common

~ 80 residues, two highly conserved regions - 4 beta sheets flanked with 2 alpha helices

premRNa processing

1. 5’ capping CTD tail

2. RNA splicing CTD tail

3. Polyadenylation CTD tail

spatial separation fo transcription and translation

protects the cell from making faulted proteins

cotranscriptional modifications

1. 7-met-guanosine coupled to 5’ end

2. methylation of ribose

3. N-methylation of adenine

phosphorylated CTD recruits cappping enzymes

1. RNA 5’ triphosphate removes the 5’ phosphate

2. guanylyl transferase attaches GMP

3. 7-methyltransferase modifies the terminal guanosine

4. mRNA 5’cap released from CTD tail

Poly a tail

increase transalation efficiency by aiding ribosome recycling and mRNA circularization

polyadenylation requires

nuclear poly A binding protein Ii PABII 200-250 nucleotides

splice sites can be determined by comparing genomic dna with

cDNA or mRNA

Group I and Ii introns

self-splicing introns - RNA is plicing itself (no proteins invovled) RNA = ribozyme

most eukaryotic cells - spliceosomal splicing

splicing assisted by at least 5 small nuclear RNAs (snRNAs)

• U1, U2, U4, U5, U6

spliceosome protein complexes associated with phosphorylated CTD tail

factors invovled in splicing: helicases

assist snRNAs and Sm protiens in: spliceosomal protein-protein interaction