BIOC520: Biological Chemistry			
L-44/45: RNA, TRANSCRIPTION, RNA PROCESSING

Objectives:
1.	To understand how RNA is transcribed
2.	To compare transcription in eukaryotes and prokaryotes
3.	To understand RNA processing mechanisms 

I.	Structure of RNA (Fig. 11.27-11.28  Marks)
  A.	RNA is a polymer of ribonucleotide monophosphates
    1.	purine bases are adenine and guanine; pyrimidine bases are
cytosine and uracil
    2.	RNA molecules can have extensive secondary structure
	a.	intramolecular base pairing
	b.	regions of base pairing in RNA form an A-type double helix
	c.	many secondary structures of RNA have defined functional roles


II.	Classes of RNA molecules
  A.	messenger RNA (mRNA)
    1.	transcribed by RNA polymerase II in eukaryotes
    2.	encode proteins
  B.	ribosomal RNA (rRNA) (Fig. 11.31-33)
    1.	18 S, 28 S, and 5.8 S rRNAs are transcribed by RNA polymerase I in
eukaryotes
    2.	5 S RNA is another type of RNA associated with ribosomes but is
transcribed by RNA polymerase III in eukaryotes
    3.	rRNA serve structural and catalytic roles in ribosomes
  C.	transfer RNA (tRNA) (Fig. 11.34-35)
    1.	transcribed by RNA polymerase III in eukaryotes
Note: all three types of RNA's above are transcribed by the same RNA
polymerase in prokaryotes
  D.	numerous other small RNA's are also found in cells--in eukaryotic
cells these can be put into two general classes:  snRNA = small nuclear
RNA & scRNA = small cytoplasmic RNA
    1.	snRNA's and scRNA's are found complexed with proteins and carried
a variety of cellular functions (snRNP and scRNP)
    

III.	Transcription (Fig. 13.1 to 13.7 and 29.10 and 29.12  Voet)
  A.	RNA molecules are transcribed from a DNA template by RNA
polymerases
  B.	requirements for RNA polymerases: DNA template, ATP, GTP, CTP,
UTP, Mg++ (no primer is necessary for RNA polymerase)
  C.	chain growth is from 5' to 3'
  D.	General mechanism of transcription


IV.	RNA synthesis in bacteria (Fig. 29.10 and 29.12; 13.6 and 13.7)
  A.	Steps of transcription 
    1.	Initiation
	a.	RNA polymerase binds to DNA and migrates to the promoter
	  1.	a promoter is a specific DNA sequence that contains a site
for transcriptional initiation--E. coli promoter contain a -10 region and
a -35 region that are important in binding polymerase 
	  2.	specific interaction between the promoter and RNA
polymerase  requires sigma factor
	  3.	initial complex between polymerase and promoter is called
the closed complex
	b.	RNA polymerase unwinds ~18 bp forming an open complex
	c.	first nucleotide, which is almost always a purine, interacts with
the open complex by binding to polymerase and base pairing with the
complementary nucleotide in the template strand 
    2.	Elongation
	a.	the second nucleotide binds to the polymerase-template complex and
a phosphodiester bond is formed
	b.	sigma factor is released and polymerase moves down the template,
unwinding the template and catalyzing the addition of each successive
nucleotide
	c.	approximately 12 nucleotides of the growing RNA chain are base
paired with the DNA template during the elongation phase
    3.	Termination (Fig. 29.15)
	a.	Termination requires special termination signals
	  1.	rho-independent termination
	  2.	rho-dependent termination 
  B.	Inhibitors of bacterial RNA synthesis
    1.	actinomycin D--binds to DNA by intercalating between base pairs
    2.	rifampicin (rifamycin)--binds to the beta subunit of bacterial RNA
polymerase
	-also inhibits mitochondrial RNA polymerase
	-used to treat tuberculosis which is resistant to most other antibiotics


V.	RNA synthesis in eukaryotes (Fig. 13.5, 13.6, 13.8, 13.13, 13.14,
13.16)
  A.	transcription and processing of mRNA
    1.	most transcription units have exons and introns
    2.	Initiation of transcription by RNA polymerase II
	a.	polymerase II does not recognize a specific sequence in the
promoter; it is positioned at the correct site by interaction with
transcription factors
	b.	many genes contain a sequence called the TATA box approximately 30
bp upstream from the transcriptional start site; a transcription factor
named TFIID specifically binds to the TATA box to help position the
polymerase at the initiation site. Some genes lack a TATA box but also
utilize a sequence-specific transcription factor to target polymerase to
the initiation site.
    3.	Capping of the 5' end of the transcript (Fig. 13.11 and 29.33)
	a.	very soon after the transcript is initiated a 7-methylguanosine
"cap" is added to the 5' end of the transcript		
	b.	7mG cap is an important signal for the translation process and may
also help protect  the message from degradation
    4.	polyadenylation (Fig. 13.12)
	a.	the 3' end of (most) transcripts is modified by polyadenylation
	b.	the poly A tail is usually about 200-250 nucleotides
	c.	the poly A tail is not encoded by the gene but is added
post-transcriptionally
	d.	polyadenylation requires a specific sequence (AAUAAA) in the mRNA
	e.	no specific termination signal is known for eukaryotic genes and
transcription proceeds past the polyadenylation signal
    5.	mRNA splicing (Fig. 13.13, 13.14, 13.15)
	a.	intron sequences are removed from primary transcripts by a process
called splicing
	b.	splicing requires consensus splicing signals at the 5' and 3' ends
of the intron, a consensus branch point 10-40 bases upstream of the 3' end
of the intron
	c.	splicing is carried out by a large complex called the
spliceosome--the spliceosome is assembled from small nuclear
ribonucleoprotein (snRNP's)
  B.	Synthesis and processing of rRNA in eukaryotes (Fig. 13.19, 13.20)
    1.	rRNA is synthesized in the nucleolus by RNA polymerase I
    2.	genes for rRNA's are found in multiple copies in tandem arrays on
the chromosome
    3.	all three major ribosomal RNA's (28S, 18S, 5.8S) are synthesized
as part of the same precursor transcript which is processed by a series of
cleavage steps to produce the mature rRNA's
    4.	assembly of rRNA's and ribosomal proteins into large and small
ribosomal subunits also takes place in the nucleolus

  C.	Synthesis and processing of tRNA (Fig. 13.21, 13.22, 13.23)
    1.	synthesized by RNA polymerase III in eukaryotes
    2.	primary transcript is a precursor that generally has extra
nucleotides on both the 5' and  3' ends; some tRNA genes also have introns
but splicing is by a completely different mechanism than with mRNA
    3.	the extra nucleotides are removed from the ends and then 3
nucleotides (CCA, these are not encoded by the gene) are added to the 3'
end post-transcriptionally
    4.	the bases of tRNAs undergo extensive post-transcriptional
modification, up to 10 % of the nucleotides can be modified
    5.	mature tRNAs have extensive secondary and tertiary structure that
is important for their function