1. bacteria "ready to go"
    -polycistronic (mult proteins)
    - no 5' modification PPP structure (no CAP)
    -internal ribosome binding sites-Shine Dalgarno seqs.
    -no poly A tail

2. Eukaryotic
    -monocistronic
     -5' 7-methyl G CAP-ribosome binding site (6-26)
    -no internal ribosome binding site
    -poly A tail 100-200
    -extensive 3' untranslated region

Eukaryotes: A long primary transcript is synthesized from the promoter
primary transcript = introns and exons and non-coding seqs
 

RNA is modified extensively in the nucleus! .....it has to be!

RNA processing...its about more than just splicing!

RNA processing (Fig 8-56):

1. splicing
2. 5' CAP
3. 3' Poly A

Modification of primary transcript occursduring elongation by enzs associated with pol II (Fig 8-49)

1. 5' CAP (Fig 8-48)
    a) added by Capping ENZ (complex)
            -5' to 5' linkage to 7-methyl G + charged
            -occurs immediately after elongation (Fig 8-49)
            -associated with pol II!

    b) Functions:
            -ribosome binding
            -stabilizes RNA
                        uncapped = degraded & retention in the nucleus
            -only pol II transcripts

2. Poly A addition

    a) Nuclease and poly A polymerase
            -bound to pol II during elongation
            -Nuclease recognizes AAUAAA in primary transcript
            -poly A polymerase adds 100-200 A's

    b) Functions:
            -Stabilizes RNA...poly A=stable
            -aids in transport out of nucleus
            -aids in translation!..interacts with CAP
                    (Fig 9-87B)

3. RNA splicing:  joining of 3' of exon 1 with 5' of exon two ........result: remove introns

split genes -R-looping using EM (Fig 8-50)

Transcript elongation and processing is continuous (Fig 8-52)

Primary Transcript "coated" with hnRNPs  heterogeneous nuclear ribonucleoprotein particles
    -prevents folding over?
    -protect emerging RNA strand
    -similar to...
      -nucleosome "like"
remember introns are 1-50kb away!

Splicing Occurs during transcript elongation (Fig 8-52)
        -splicing factors are found associated with some forms of RNA polymerase!!

Mediated by riboprotein complexes snRNPs  small nuclear ribonucleoproteins U1-U12
    -protein
    -RNA...used for seq recog' (basepairing)
            recog' "splicing sites"

The spliceosome: (Fig 8-52 and Fig 8-54)
        -SNURPs
        -non-SNURP proteins
        -forms during elongation

Splicesome formed by protein-RNA and RNA-RNA basepairing interactions with conserved seqs in intron

if conserved and found in exon.....

Directed by specific (conserved) seqs  (Fig 8-53) thalassemia
    -5' splice site (donor)
    -internal branch point "A"
    -catalytic "residue"
    -3' splice site (acceptor)

Spliceosome function: bring 5' and 3' seqs together! (Fig 8-54)

1. Snurps bind to 5' and 3' splice sites: "protein bridge" 10-50 kb away
2. Activation of branch point A and catalytic attack of 5' site
3. Formation of "lariat"
4. cleavage and 3' splice site and ligation of exons
5. lariat removal and degradation

Stepwise addition and activation of multi-protein-RNA complexes  iniated on conserved nucleotide elements.....Sound familar?

Very efficient and FAST in vivo!
    -no intermediates!
    -difficult to detect primary transcript
    -"always" pairs the two closest exons

why?....emerging transcript (Fig 8-52)

Self-catalyzed RNA splicing-no proteins

First: tetrahymena and T4 bacteriophage rRNA (Fig 3-21, mitochondria and chloroplasts too!

Two classes: (Fig 8-58)

Group I:
    -reactive "G" in intron
    -no lariat, direct cleavage and ligation

Group II:
    -reactive "A" in intron
    -lariat formation
    -precursor to spliceosome mechanism

Self-splicing group II introns: precursor to splicisome-mediated
    -same pH, ion requirements as Spliceosome mediated splicing
    -same chemical rxn. Proteins just structural to accommodate large introns and regulated splicing
    -RNA structure subs' for proteins (Fig 3-24)
    -All "info" in nucleotide seq

Need for spliceosome?:
    -large introns
    -coprocessing
    -regulation