Structure of Prokaryotic mRNA
Shine-Dalgarno sequence: the site where ribosomes bind to the mRNA; 7 nts upstream of the start codon
- Specifies the amino acid sequence
- -i.e. where the codons are found
Prokaryotic mRNA CAN contain more than 1 coding region
a polycistronic ('multigenic') RNA
one promoter -> mRNA -> multiple polypeptides
Eukaryotes have mostly monocistronic RNAs
- 1) Involves MORE enzymes & factors
- 2) Occurs even though the DNA is packaged into NUCLEOSOMES
- 3) Is physically SEPARATE from all translation activities
Three RNA Polymerases in Eukaryotes
RNA Polymerase I (Large rRNAs), RNA Polymerase II (pre-mRNA, some snRNAs, snoRNAs), RNA Polymerase III (tRNAs, small RNA, some snRNAs)
RNA Polymerase II is responsible for txn of MOST eukaryotic genes
THREE Stages of Transcription
- 1) Initiation
- 2) Elongation
- 3) Termination
Eukaryotic promoters can consist of MANY RECOGITION SEQUENCES for transcription factors
The two most commonly found consensus sequences are the GC box and the TATA box
DO NOT Confuse DNA & Protein
DNA sequences - 'sequence' 'promoter' 'box' 'element'
- 'factor' 'activator' 'repressor'
Promoter recognition in eukaryotes is carried out by accessory proteins, that in turn recruit RNA polymerase
- General Transcription factors (GTFs)
- ex: IID, IIA, IIB, etc.
Together they comprise a 'basal txn apparatus'
Formation of the Basal Apparatus
1) Transcription factor IID = TFIID, binds the TATA box sequences
TFIID is a holoenzyme with several subunits including TATA binding protein = TBP
2) RNA poly II, other GTFs & a mediator complex bind to TFIID at the promoter
3) Interactions b/w REGULATORY PROTEINS bound to upstream regulatory sequences and the BASAL APPARATUS influences LEVELS of txn.
The activity of the basal apparatus is influenced by upstream regulatory elements
The regulartory promoter of a gene can contain MULTIPLE ELEMENTS outside of the core promoter
Some regulatory elements functions at a DISTANCE from the core promoter, and can work in BOTH DIRECTIONS!
Formation of the Basal Transcription Apparatus
When all factors are present, the DNA is UNWOUND and RNA Pol starts making RNA from +1
RNA Pol II and some associated factors MOVE DOWN the DNA making a pre-mRNA
The Pre-mRNA is 'Capped'
* A 7-methyl guanosine (7MG) is added. 5'-5' phosphate bond
* Occues EARLY in elongation
* Methyl groups (-CH3) are often added to the 2'-OHs of the next few nucleotides
1) PROTECTS tge end of the mRNA from degradation
2) Is required for TRANSLATION
1) RNA pol II transcripts are CUT downstream of a conserved sequence.
2) A poly(A) polymerase adds a long run of AMPs onto the 3' end of the pre-mRNA
THREE Modifications to Pre-mRNAs
- 1) Addition of 5' cap (7MG)
- - shortly after initiation
- - stability
- 2) Addition of 3' poly-A tail
- - after termination
- - stability
- 3) Removal of intervening sequences
- - RNA splicing
- - after termination
-Most eukaryotic genes containing BLOCKS of coding sequence (exons) and non-coding sequence (introns)
-Both are present in the initial RNA transcript, but the introns are removed before export
How were INTRONS discovered?
Proof that introns exist came from DNA:RNA hybridization experiments
HOW is RNA splicing accomplished?
- - Must be PRECISE, to maintain the sequence of the polypeptide
- - requires consensus sequences:
- - at exon/intron JUNCTIONS
- - WITHIN the intron
Intron Consensus Sequence
- - Short consensus sequences at each splice junctions
- - A poorly conserved sequence WITHIN THE INTRON upstream (18-40 nts) of the 3' splice site
RNA splicing occurs through a 'lariat' intermediate
- - includes FIVE small nuclear ribonucleoprotein particles (snRNPs)
- - they contain FIVE snRNAs (approx. 1-200 nts): U1, U2, U4, U5, U6
- - snRNAs recognize mRNA splice sites by complementary base pairing!
- - snRNAs also have catalytic activity
Mechanisms of Splicing
- 1) the 5' splice site is cut
- - exon 1 is held in place by the spliceosome
- - 5' end of intron attaches to branch point
- 2) The 3' splice site is cut
- - Exon 1 is joined to Exon 2 by a phosphodiester bond
- - the lariat is released
Some genes are spliced in DIFFERENT ways, producing DIFFERENT mRNAs!
Occurs SPATIALLY and TEMPORALLY
Alternate 3' cleavage can produce DIFFERENT RNAs from ONE gene.
Non - mRNA May Be Spliced By Other Mechanisms
- Some RNA molecules can self-splice (autocatacytic activity)
- - Exs:
- *Some ribosomal RNAs
- * Some chloroplast and mitcochondrial RNAs
Transfer RNA (tRNA)
- - short chains (70-90 nucleotides long)
- - have special secondary structure
- - 'link' the mRNA to the polypeptide (crick's hypothesis)
- - all tRNAs (proks/euks) are processed
- - have more modified bases than any other class of RNA
- - tRNA genes may be present in numerous copies
- Some RNA sequences are CHANGED after transcription!
- - some bases are changed
- - Some bases are CHANGED
- - Some bases are ADDED or DELETED
- - CONTRADICTS central dogma
- RNA editing
tRNAs are also Spliced
- - by distinct splicing enzymes
- - an intron in a pre- tRNA is CUT OUT, then the ends are LIGATED
- - uses TWO enzymes
All tRNAs are EXTENSIVELY PROCESSED to form the final functional molecules!
Where do rRNAs come from?
Genes can be present in MULTIPLE COPIES
in euks, made in the nucleolus
- Several different ones:
- - proks: 3 rRNAs
- - Euks: 4 rRNAs
Also undergo PROCESSING
Involves small nucleolar RNAs (snoRNAs)