Chuck C.-K. Chao (趙清貴)Tumor Biology Laboratory

Department of Biochemistry & Molecular Cell Biology& Graduate Institute of Basic Medical Sciences

Chang Gung University

Tel: 03-3283016 ext. 5157, 5151E-mail: cckchao@mail.cgu.edu.tw

RNA Metabolism

• DNA-dependent synthesis of RNA

• RNA processing

• RNA-dependent synthesis of RNA & DNA

RNA (Ribonucleic Acid)

Transcription: an enzyme system converts the genetic information in dsDNA into an RNA strand with a base sequence complementary to one of the DNA strand.

• messenger RNA (mRNA)

• transfer RNA (tRNA)

• ribosomal RNA (rRNA)

RNA Is Synthesized by RNA Polymerase

Transcription in E. coli

• encompasses ~35 bp of DNA (revealed by footprinting, p.985)

• requires DNA template, NTP & Mg2+

• adds nucleotide units to the strand’s 3’-OH end in 5’ 3’ direction

~17 base pairs of DNA template are unwound

Supercoiling of DNA brought about by transcriptionPositive supercoils form ahead of the transcription bubble,

and negative supercoils form behind.

The coding strand for a particular gene may belocated in either strand of a given chromosome. e.g., adenovirus genome (36,000 bp)

Many of the mRNA are initialy synthesized as a long transcript(25,000 nt), which is then extensively processed to producethe separate mRNA.

Structure of E. coli RNA polymerase

Lacks 3’ 5’ exonuclease activityerror: 10-4 to 10-5

“holoenzyme”

RNA Synthesis Is Initiated at Promoters

Consensus sequence of typical E. coli promotersrecognized by RNA polymerase holoenzymecontaining 70

RNA Polymerase Leaves Its Footprint on a Promoter

“Footprinting”:a method that provides informationabout the interaction betweenRNA polymerase and promoters.

Specific Sequences Signal Termination of RNA Synthesis

• Not yet well understood in eukaryotes

• At least two signals in E. coli: (rho)-dependent and -independent

-independent termination of transcription

Eukaryotic Cells Have Three Kinds of Nuclear RNA

Polymerase

• RNA polymerase I: rRNA

• RNA polymerase II: mRNA etc.

• RNA polymerase III: tRNA, 5S rRNA etc.

Common Sequences in Promoters Recognizedby Eukaryotic RNA Polymerase II

“Initiator sequence”

RNA Polymerase II Requires Many Other Protein Factors for Its Activity

Transcription at RNA Polymerase II Promoters

• assembly• initiation• elongation• termination

The Structure of TBP (gray) Bound to DNA (blue and white)

RNA Polymerase Can Be Selectively Inhibited

Inserted into DNAbetween G/C

• actinomycin D: prok/euk.• rifampicin: prok.• -amanitin: euk. pol II etc.

G

C

A Complex of Actinomycin D and DNA

RNA Metabolism

• DNA-dependent synthesis of RNA

• RNA processing

• RNA-dependent synthesis of RNA & DNA

Maturation of mRNA In a Eukaryotic Cell

Phillip Sharp & Richard Roberts, 1977The genes for polypeptides in eukaryotes are often interrupted by noncoding sequences (introns).i.e., “split gene”

e.g., chicken ovalbumin gene

intron: A-Gexon: 1-7

Chicken ovalbumin gene

Introns are removed by splicing

Introns

• Group I: guanosine 3’OH as nucleophile

• Group II: adenosine 2’OH in intron as nucleophile

• Group III: dependent on snRNPs, pronounced “snurps” (small nuclear ribonucleoproteins), not self-splicing

• Group IV: need ATP and endonuclease

RNA Catalyzes Splicing

Thomas Cech et al., 1982 (p.994)protozoan Tetrahymena thermophilathe splicing mechanism of group I rRNA intron

Sidney Altman et al., 1983 (p.1004)E. coli M1 RNA (377 nt) of RNase P cut tRNA

Transesterification reaction: the first step in the splicing of group I introns

Splicing mechanism of group I introns

Splicing mechanism of group II introns

“lariat”

Splicing mechanism of group III intronsin eukaryotic mRNA primary transcripts

snRNAs (small nuclear RNAs)

Assembly of spliceosomes

snRNPs (“snurps”) = snRNA-protein complexes

Splicing mechanismof group IV intronsin yeast tRNA

Eukaryotic mRNA Undergo Additional Processing

• adding 5‘ cap

• adding poly(A) tail

7-methylguanosine is added to the 5’ end of almost all eukaryotic mRNAsin 5’,5’-triphosphate linkage.

Methyl groups (red) are sometimes foundat the 2’ position of the first and second nt.(not in yeast)

first

second

cap

adoMet = S-adenosylmethionine

Generation of the 5’ cap

adoHcy = S-adenosylhomocysteine

Addition of the poly(A) tail to the primary RNA transcript of eukaryotes

Overview of the processing of a eukaryotic mRNA

Multiple Products Are Derived from One Gene by Differential RNA Processing

Alternative cleavage & polyadenylation

Alternative splicing

E.g., Alternative processing of the calcitonin gene transcript in rats

(calcitonin-gene-related peptide)calcium-regulatinghormone

rRNAs and tRNAs Also Undergo Processing

Processing of pre-rRNA in bacteria

Processing of pre-rRNAs in vertebrates

Processing of tRNAs in bacteria & eukaryotes

Some modified bases of tRNAs, produced in post-trancriptional reactions

Some Events in RNA Metabolism Are Catalyzed by RNA Enzymes

Hammerhead ribozyme (only 41 nucleotides)requires Mg2+

E.g., the self-splicing rRNA intronfrom Tetrahymena

Internal guide sequence (boxed)pairs with splice site at 5’ end (red arrow) &3’ end (blue arrow)

Intron (yellow)exon (green)catalytic core (shaded)

L-19 IVS is generated by the autocatalytic removal of 19 nt from 5’ end of the spliced intron.

(414 nt)

(395 nt)

L-19 IVS (intervening sequences)has catalytic activity in vitro, but quickly degraded in vivo.

RNA enzymes:L-19 IVS, from group I introns, lengthens some RNA oligonucleotides at the expense of others in a cycle of esterification reaction

Oligo C paired with the same G-rich internal guide sequences

L-19 IVS

RNA Processing

• …...• Cellular mRNA Are Degraded at Different Rates by ribonucleases usually in a 5’ 3’ direction occasionally in a 3’ 5’ direction.

In bacteria: a hairpin structure in mRNA with -independent terminator (p.986) confers stability.

In eukaryotes: the 3’ poly(A) tail confers stability.

A major pathway: shortening the poly(A) tail > decapping the 5’ end > degrading the RNA in the 5’ 3’ direction.

Polynucleotide Phosphorylase Makes RandomRNA-like Polymers

Marianne Grunberg-Manago & Severo Ochoa, 1955

(NMP)n + NDP (NMP)n+1 + Pi

RNA Metabolism

• DNA-dependent synthesis of RNA

• RNA processing

• RNA-dependent synthesis of RNA & DNA

Extension of the central dogma to includeRNA-dependent synthesis of RNA and DNA

Retroviral infection of a mammalian cell and integration of the retrovirus into the host chromosome

Reverse transcriptase

Reverse Transcriptase Produces DNAfrom Viral RNA

Howard Temin & David Baltimore, 1970

genetic information can flow “backward” from RNA to DNA

Structure and gene products of an integrated retrovirus genome

Retrovirus Cause Cancer and AIDS

Rous sarcoma virus genome

Peyton Rous: RSV from chicken sarcoma, 1911Harold Varmus & Michael Bishop: src oncogene

Retrovirus Cause Cancer and AIDS

The genome of HIV, the virus that causes AIDS

Fighting AIDS with Inhibitors of HIV Reverse Transcriptase

Many transposons, Retroviruses, and Introns May Have a Common Evolutionary Origin

Eukaryotic transposons:structurally similar to retroviruses,but lacking env gene.

Many transposons, Retroviruses, and Introns May Have a Common Evolutionary Origin

Introns that move:

Many transposons, Retroviruses, and Introns May Have a Common Evolutionary Origin

Introns that move:

Many transposons, Retroviruses, and Introns May Have a Common Evolutionary Origin

Introns that move:

Telomerase Is a Specialized Reverse Transcriptase

The internal template RNAbinds to and base-pairs withthe DNA’s TG primer

Adding more T & G

Reposition of the internal template RNA

Telomerase Is a Specialized Reverse Transcriptase

Form T loops in telomeres (~103 bp) of higher eukaryotes including mammals

How to protect ssDNA end?

By specific binding proteins in telomeres (~102 bp) of

lower eukaryotes

EM of a T loop of chromosome endfrom mouse hepatocyte

Some Viral RNAs Are Replicated by RNA-Dependent RNA Polymerase

Some E. coli RNA viruses, e.g., f2, MS2

have RNA-dependent RNA polymerase (RNA replicase)

which contains four subunits (210-kDa):

one viral replicase for replication,

three host proteins (elongation factors Tu and Ts, and

30S ribosome protein S1) for locating the 3’ends of the

viral RNA

RNA Synthesis Offers Important Cluesto Biochemical Evolution

Carl Woese, Francis Crick & Leslie Orgel, 1960stheory: RNA might serve as both information carrier & catalyst

Thomas Cech et al. & Sidney Altman et al., 1980sproof: catalytic RNAs

>> “RNA world” might have been important in the transition from prebiotic chemistry to life!

Possible prebiotic synthesis of adenine from ammonium cyanide

RNA World Hypothesis:Can a ribozyme replicate in a template-dependent manner?

The first step: making a ribozymeReversible attack of a guanosine on the 5’ splice sitein the removal of the self-splicing group I intron(i.e., ribozyme P1 region, p.1003)

RNA World Hypothesis:Can a ribozyme replicate in a template-dependent manner?

The ribozyme makes template RNA capable of further RNA polymerization reactionsIt can link oligo-RNAs in a process equivalent to the reversal reaction in (a)

The ribozymes found in nature have a limited repertoire of catalytic functions, but the catalytic potential of RNA is far greater.

Rapid search for pools of random polymers of RNAs with new catalytic functions is required!

The search for RNAs with ATP-binding functions bySELEX (systematic evolution of ligands by exponential enrichment)

25 nt oligo in maximum

425 = 1015 random RNA oligos

ATP-binding RNA oligonucleotideisolated by SELEX