4 RNA Synthesis

• 1. the pentose sugar in RNA is ribose instead of deoxyribose
• 2. RNA has Uracil (U) instead of Thymine (T)
• 3. DNA normally exists as a double helix whereas RNA exists as a single strand

• the sequence of DNA that is copied during the synthesis of mRNA
• (RNA is synthesized 5' → 3’)

• the strand with a base sequence directly corresponding to the mRNA sequence
• this sequence corresponds to the codons that are translated into protein
• also known as the mRNA-like strand, or (+) strand

• the coding strand, also called the (+) strand
• the other strand is called the template or (-) strand

• • RNA polymerase I, II & III
• • they transcribe different genes
• • are complexes made up of protein subunits
• • while some subunits are found in all 3, they also have nonidentical subunits, which explains why they’re responsible for DIFFERENT jobs

• found in the nucleOlus & transcribes 28S, 18S & 5.8S rRNAs (ribosomal RNAs)

• is important for PROTEIN SYNTHESIS

• found in the nucleus & transcribes pre-mRNA (heterogeneous nuclear RNA)

• is important for protein encoding, RNA splicing, & post-transcriptional gene control

• is incapable of locating a promoter in the absence of the basal transcription factors

• found in the nucleus and transcribes tRNA, 5S rRNA & other small RNAs

• small RNAs are involved in the splicing process

by their sensitivities to α-amanitin

• pol I: resistant
• pol II: sensitive
• pol III: intermediate

because there are so many identical subunits, it appears that many of the eukaryotic RNA polymerase components arose via gene duplication & subsequent sequence divergence

1. rRNA transcription units are arranged in multiple, tandemly arrayed copies in the genome; individual units are separated by a spacer

2. the rRNA transcription unit is transcribed as a SINGLE RNA chain that’s 13,700 nts long

3. 2 transcription factors (B & S) promote binding of RNA Pol I to the “regulatory region”

• factor B (for binding) binds to sequences within the regulatory region

• its binding is greatly stimulated by a 2nd factor called the S factor

• upon assembly of these 2 factors, RNA pol I can bind & transcription begins

• (transcription terminates past the 28S rRNA near the next start site in the array)

a string of T residues

transcription factors B & S (B for binding, S for stimulating)

they help build ribosomes (structural) & have catalytic activity

60S & 40S

the 28S & 5.8S rRNA associate with the 60S ribosomal subunit

the 18S associates with the 40S ribosomal subunit

RNA Polymerase III [eg. tRNAs, RNAs that assemble in small nuclear ribonucleoproteins (snRNPs), 5S rRNA & many others whose functions are not known]

• made up of 120 nucleotides & is a component of the large ribosomal subunit

• the activation region for 5S rRNA lies WITHIN the gene

• assembly factors TFIIIA & TFIIIC bind to this activation region & enable the binding of TFIIIB (a transcription factor)

• RNA pol III recognizes TFIIIB (+ other factors) → transcription begins

• termination occurs at a string of T residues

• the primary transcript is processed by removal of several bases from the 3′ end (other Pol III transcripts, eg. tRNA, are more extensively processed)

Within the genes!

TFIIIA, TFIIIB & TFIIIC (the Roman numeral in the name of the tx factor tells you which pol it’s associated with)

• TFIIIA & TFIIIC bind to the activation region located IN the gene

• this recruits TFIIIB to bind which subsequently recruits RNA pol III

• termination occurs at a string of T residues

regions of DNA where RNA polymerase binds to initiate transcription

crucial to the accuracy & rate of transcription initiation

are usually located immediately upstream of the gene

upstream: OPPOSITE to the direction of transcription

downstream: in the SAME direction as transcription

• sequences of DNA that can up-regulate transcription of neighboring genes
• - they exert their effects over long distances, function in either orientation & are located upstream, downstream or in a given gene

the TATA box - directs RNA pol II binding, found ~25 bases upstream of the gene (-25)

is HIGHLY conserved

basal transcription factors assemble

specifically, TATA-binding protein (TBP), a subunit of TFIID, binds

transcription factors required for the initiation of transcription (DNA → RNA) in all genes

• in response to injunctions from activators, these factors position RNA pol II at the start of the protein-coding region of a gene

• RNA pol II is incapable of locating a promoter without basal transcription factors

TFIID, a subunit of which is TBP (TATA-binding protein)

TFIID phosphorylates pol II, activating it

a basal transcription factor that interacts with TFIID in addition to binding to pol II & help direct it to the promoter

• sequences of DNA that can up-regulate transcription of neighboring genes

• can exert their effects over long distances & function in either orientation

• are located upstream, downstream or within a given gene

• TFs that bind to enhancers are called Activators

(VOLUME CONTROL)

the NUCLEUS

1. 5' 7-Methylguanylate CAP

2. 3' poly-A tail [a feature of RNA pol II transcripts only]

3. intron splicing, exon ligation

7-methylguanylate (m7G) cap

• it prevents the RNA from being degraded & later signals for translation to occur

• it’s attached via a 5' → 5' triphosphate bridge

a ~250 nt poly-A tail

• it’s not placed where the mRNA transcript has stopped being made, it’s added UPSTREAM of the end of the entire primary transcript

• there’s a cleavage event & then the poly A tail is added

AAUAAA

located 10-30 nucleotides upstream of the site of cleavage & subsequent polyadenylation

recognizes the AAUAAA sequence on pre-mRNA & recruits poly(A) polymerase

• upon cleavage, poly(A) polymerase adds ~250 adenylate residues

a polymerase that adds the poly-A tail without using a template

• poly-A tails are NOT encoded within genes

the 5’ cap, THEN the polyA tail is added

part of primary transcript but not found in mature mRNA

• variable both in number (eg. 2 in β-globin & 50 in collagen) & length (up to ~200 kb)

the part of a eukaryotic gene coding for RNA which will ultimately be spliced into functional mRNA

• are thought to contain individual functional domains

the process by which introns are removed from the pre-mRNA molecule

a large ribonucleotide protein complex similar to the ribosome that contains smaller nuclear snRNPs (U1, U2, U4, U5 & U6) that aid in the removal of introns from pre-mRNA molecules

splicing takes place on the spliceosome

• GU residues at their 5' end

• AG residues at their 3' end

• a pyrimidine-rich region just upstream of the 3' splice site

cleavage of the 5' exon-intron junction

1. cleavage at the 5’ splice site → formation of a lariat

2. cleavage of the RNA at the 3’ splice site with concomitant joining of the two exons

3. the 3' intron-exon junction is cleaved & the neighboring exons are ligated together

the circularization of the intron with a Branch Point between the 5' G residue linked (5' to 2') to an Adenosine residue located near the 3' end of the intron

U1 → 5' exon-intron junction

U2 → branch point (GU → A)

U5 → binds just upstream of the 3' intron-exon junction

(Several additional snRNPs are required to form a spliceosome)

• characterized by defective synthesis of 1 or more hemoglobin chains as a result of mutations that prevent proper intron splicing

• hereditary abnormalities of hemoglobin production in which there is a quantitative deficiency of either β-globin (β- thalassemia) or α-globin (α-thalassemia)

a single pre-mRNA can be spliced in different ways, yielding different mature mRNAs; a single gene can code for a series of related, but non-identical proteins

• accounts for much of our diversity

eg. alpha-tropomyosin (muscle contraction protein)

Alternative Splicing

soluble proteins lack two exons that encode very hydrophobic amino acids that normally anchor the membrane bound form on the cell surface

1. Rifampicin: blocks initiation of bacterial RNA synthesis (inhibits bacterial RNA pol)

2. Actinomycin D: binds to dsDNA & prevents it from being used as a template (inhibits mammalian RNA pol I)

• Primary Transcript: RNA that has both introns & exons

• cDNA: DNA copy of mRNA, therefore is missing introns & includes polyA tail + post-trascriptional modifications

• used to clone individual mRNA molecules

1. start with cells that express the gene of interest

2. isolate mRNA using oligo (dT) columns to selectively remove poly A containing mRNA

• 3. mix isolated mRNA with oligonucleotides containing ~20 deoxythymidines
• - these oligonucleotides act as primers for reverse transcriptase

• 4. reverse transcriptase catalyzes the synthesis of a DNA copy of the mRNA molecule
• - end up with an RNA-DNA hybrid

5. reverse transcriptase is over zealous & begins to copy the newly synthesized DNA strand

6. this material is used by a DNA pol to synthesize the second strand

7. hairpin loop is cleaved by S1 nuclease, a single strand specific nuclease

8. cDNA can be ligated into a suitable vector (either plasmid or phage)

• 9. cDNA containing vector is then introduced into bacterial cells
• - plasmid vectors frequently contain a gene that confers resistance to ampicillin; bacteria lacking the plasmid do not survive antibiotic selection

10. resulting collection of cloned cDNA fragments propagated in bacteria is called a cDNA library

False. They generally contain a GC-rich region instead.

FALSO, only those RNAs that are to become mRNAs acquire a poly(A) tail

GU, AG

18S rRNA

28S, 5.8S & 5S rRNAs

5S rRNA (it is synthesized by RNA poly III)