Transcription and Splicing

Gene expression

The "Central Dogma" of molecular genetics: the flow of genetic information is from DNA to RNA to protein

Gene expression involves two fundamental processes: Transcription/RNA synthesis & translation/protein synthesis

DNA, RNA and protein synthesis have similar and distinct features

Features common to transcription, translation, and replication:

Template dependence:

DNA (for replication and transcription)
RNA (for translation)

Processive and unidirectional:

5' to 3' (DNA/RNA); N- to C-terminal (proteins)

Molecular recognition based upon base-pairing:

A-T and G-C for DNA synthesis
A-U and G-C for RNA synthesis
codon (in mRNA) and anticodon (in tRNA) for translation

Energy consuming (dNTP's and/or NTP's)

Differences

Enzymatic machinery:

Polymerases: DNA and RNA
Ribosomes + assoc. factors (IF's/EF's)

Polarity of template (i.e., reading orientation):

Replication/transcription: DNA template is read from 3' to 5'
Translation: mRNA is read from 5' to 3'

Template/product "stoichiometry"

1 nucleotide : 1 nucleotide (DNA+RNA synthesis)
3 nucleotides (a codon) : 1 amino acid (protein synthesis)

Subcellular localization (eukaryotes/nuclear genome):

Replication and transcription: nucleus
Translation: cytosol

Initiation/Termination signals (in template):

DNA: initiates at replication origins (lagging strand "initiates" at multiple RNA primers)
RNA: promoters and terminators/ poly-A sites
Protein: Start codons (AUG=Met) and Stop (UAA, UAG, UGA) codons

Ribonucleic acid

RNA is (usually) single-stranded:

Only one of the two DNA strands is transcribed (for any given gene)

Does not (necessarily) obey Chargaffs rules

Secondary structure: ssRNA may form double helical regions

hairpins, stem-loops, etc.
complementary regions result from palindromes/ inverted repeats (in duplex DNA sequence)

Certain RNA molecules fold into complex structures with catalytic activity (ribozymes)

Protein synthesis involves 3 major classes of RNA:

1) mRNA: contain sequences that will be translated into proteins

2) tRNA: linked to specific amino acid that will be added to growing polypeptide chain

3) rRNA: play key structural and functional roles in the ribosome

Other classes of "functional RNA" participate in splicing (snRNA), RNA modification, etc.

All three classes are encoded by corresponding genes

In eukaryotes, tandemly-repeated ribosomal RNA genes (rDNA) comprise the nucleolus organizer (NO)
Protein coding (mRNA) genes are most numerous and diverse

Major differences between prokaryotic and eukaryotic mRNAs:

Prokaryotic mRNAs are often polycistronic: code for multiple polypeptides
Eukaryotic mRNA is capped (5' end) and polyadenylated (3' end)
Eukaryotic mRNAs are generated by splicing of pre-mRNA

Transcription/RNA synthesis

RNA polymerase:

Adds ribonucleotides to 3' end of growing RNA chain (using rNTP precursors)
Uses the "non-coding" DNA strand as a template in order to transcribe the "coding" (non-template) strand
Primer-independent: can initiate RNA synthesis de novo
Requires accessory protein(s) to "find" promoter/start site: sigma subunit (E. coli) or "transcription factors" (eukaryotes)

RNA polymerase diversity:

Prokaryotes: "holoenzyme" consists of common "core" enzyme (alpha₂beta beta') plus a variable sigma subunit (but usually sigma⁷⁰)
Eukaryotes have three distinct nuclear enzymes (I, II, and III) that:

Transcription involves three distinct phases: initiation, elongation, and termination

Initiation and termination require distinct "signals" (sequences) within the gene

Gene regulation may involve all three phases, but initiation is the most important

Transcription initiation

Promoters:

DNA sequences used to direct RNA polymerase to the transcription start site
Used to orient polymerase: allow selection of correct strand
Promoter function usu. involves specific "consensus sequences" in vicinity of start site
"Core" promoter elements are bound ("footprinted") by RNA polymerase (E. coli holoenzyme) and/or general/basal transcription factors such as TFIID (eukaryotes)
Transcriptional regulation or enhancement may be conferred by additional sequences outside of the promoter

Initiation in prokaryotes (E. coli):

consensus sequences at -10 (Pribnow box) and - 35 are recognized by sigma subunit
(additional upstream sequences that bind other factors may enhance transcription of certain genes)
polymerase-bound complex is converted from "closed" to "open" when DNA melts
RNA polymerase initiates synthesis at start site using single-stranded portion of "non-coding", or "template" strand
Sigma subunit dissociates during elongation

Termination of transcription

Terminators in prokaryotes:

Polymerase stops elongation and dissociates from template
Rho-independent terminators (most common): stem-loop followed by poly-U at 3' end of RNA
Rho-dependent termination catalyzed by rho protein; poly-U and stem-loop not required

Eukaryotes:

Poly-A tail is added to 3' end of mRNA ~ 20 bases downstream of polyadenylation signal (AAUAAA)
Tails vary in length (20-200 bases) and are not present in the DNA sequence
Polymerase transcription continues far downstream of polyadenylation site
Tailing requires specific nuclease (RNA cleavage) and a template-independent, poly-A polymerase

RNA Splicing (Eukaryotes)

Splicing occurs in nucleus: primary transcripts = hnRNA (heterogeneous nuclear)

Most splicing involves snRNPs

snRNPs are complexes between snRNA and specific proteins
U1 and U2 recognize the 5' splice site and branchpoint, respectively
Recruitment of additional snurps generates a catalytically active "spliceosome"
Intron is removed as a "lariat": 5' end is linked by phosphodiester bond to 2' -OH of nucleotide at branchpoint

Consensus sequences for spliceosome-mediated splicing fall mainly within the intron:

5' splice site
branchpoint
3' splice site
spliceosome structure/assembly involves RNA/RNA complementarity

Spliceosomes are assembled co-transcriptionally

Introns appear to be removed in a preferred sequence

Alternative splicing can give multiple transcripts from one gene:

an exon may have two alternate splice junctions
an exon may be skipped entirely (in some mRNAs)
may be used to generate distinct protein isoforms
may be used to regulate gene expression

Mutations in splice sites can disrupt gene function (e.g., thalassemia)