BIOL 200 (Section 921) Lecture # 4-5, June 22/23, 2006 UNIT 4: BIOLOGICAL INFORMATION FLOW - from DNA (gene) to RNA to protein Reading: ECB (2nd Ed.) Chap 7, pp. 229-262; Chap 8, pp. 267-286 (focus only on parts covered in lecture). Related questions 7-8, 7-9, 7-11, 7-12, 7-14, 7-16, 7-17, 8-5a,b,d; or ECB (1st ed.) Chap 7, pp. 211-240; 263-4; Chap 8, pp. 257-270 (focus only on parts covered in lecture). Related questions 7-9, 7-10, 7-12, 7-13, 7-15, 7-18, 7-19; 8-7b,c,e.

Lecture # 4,5: Learning objectives Understand the structural differences between DNA and RNA, and the directionality of transcription. Understand the structure of a eukaryotic gene. Describe the general mechanism of transcription, including binding, initiation, elongation and termination. Discuss factors regulating transcription. Describe processing for all three types of RNA, and discuss why it must occur. Describe the structural features of ribosomes. Describe the genetic code and how it is related to the mechanisms of transcription and translation. Describe the coupling of tRNA and discuss the specificity of amino acid tRNA synthetases. Describe the general mechanism of translation including binding, initiation, elongation and termination.

Genetic information flow [Fig. 7-1] Transcription-same language (nuc. to nuc.) Translation-different languages (nuc. to protein)

Eucaryotic vs. Procaryotic Biol. Info Flow [Fig. 7-20]

DNA vs. RNA: Differences in chemistry [Fig. 7-3]

DNA vs RNA (Lehninger et al. Biochemistry)

DNA vs. RNA: Intramolecular base pairs in RNA [Fig. 7 DNA vs. RNA: Intramolecular base pairs in RNA [Fig. 7.5] and Intermolecular base pairs in DNA [Fig. 5-7] assist their folding into a 3D structure RNA DNA

Procaryotic vs. eucaryotic transcription [Fig. 7-12] Fig. 7-12, p. 219

In procaryotes, clusters of genes called operons can be carried by one mRNA [Fig. 8-6]

Fig. 7-9:Parts of a gene [Fig. 7-9] Transcription start site Promoter sequence terminator DNA template

Transcription factor binding to DNA [Fig. 8-4] What types of bonds can you identify between this DNA and protein?

Eukaryotic transcription factors promote RNA Polymerase II binding [Equiv. Fig. 8-10, 2nd Ed.]

Directions of transcription along a short portion of bacterial chromosome [Fig. 7-10, p. 218] 3’ 5’ 3’ 5’ DNA always read 3’ to 5’! RNA always made 5’ to 3’!

RNA Polymerase: (1) Unwinds and rewinds DNA; (2) Moves along the DNA one nucleotide at a time; (3) Catalyzes The formation of phosphodiester bonds from the energy in the Phosphate bonds of ATP, CTR, GTP and UTP.

Transcription by RNA polymerase in E. coli [Lehninger et al Transcription by RNA polymerase in E. coli [Lehninger et al. Principles of Biochemistry]

introns exons Fig. 7-37 preRNA DNA in nucleus 5’ cap splicing polyA tail mature mRNA

Fig. 7-12: mRNAs in eukaryotes are processed Prokaryote mRNA Eukaryote mRNA 3’ tail 5’ cap

Fig 7-12B 5’ cap Functions: - Increased stability Facilitate transport mRNA identity

Bacterial vs. Eukaryotic genes [Fig. 7-13]

RNA splicing removes introns [Fig. 7-15] RNA splicing: (1) done by small nuclear ribonuclear protein molecules [snRNPs, called as SNURPs] and other proteins [together called SPLICEOSOME], (2) Specific nucleotide sequences are recognized at each end of the intron

Fig. 7-17 RNA Splicing by SnRNP+proteins=spliceosome 5’splice site snRNP 3’ splice site exon1 intron lariat formation

Fig. 7-17 RNA Splicing Lariat=loop of intron Spliceosome Exons joined Mature mRNA exons joined together-introns removed

Removal of intron in the form of a lariat [Fig. 7-16]

Detailed mechanism of RNA splicing [Fig. 7-17]

Is RNA splicing biologically important or a wasteful process? Pre-mRNA can be spliced in different ways (alternate RNA splicing), thereby generating several different mRNAs which code for several different proteins [e.g. approx. 30,000 human genes can produce mRNAs coding for more than 100,000 proteins] Introns quickens the evolution of new and biologically important proteins e.g. three exons of the β-globin gene code for different structural and functional regions (domains) of the polypeptide

Alternative splicing can produce different proteins from the same gene [Fig. 7-18]

Becker et al. The World of the Cell

Transcription of two genes seen be EM [Fig. 7-8]

Becker et al. The World of the Cell

Biological information flow in procaryotes and eukaryotes [Fig. 7-20]

Specialized RNA binding proteins (e. g Specialized RNA binding proteins (e.g. nuclear transport receptor) facilitate export of mature mRNA from nucleus to the cytoplasm [Fig. 7-19]

rRNA processing [Fig. 6-42 in Big Alberts] 45S transcript 18S 28S 5.8S 5S made elsewhere Small ribosomal subunit Large ribosomal subunit

Processing of transfer RNA (tRNA) [Becker et al. The World of the Cell]

From RNA to Protein: translation of the information Translation: conversion of language of 4 nucleotides in mRNA into language of 20 amino acids in polypeptide Genetic code: a set of rules (triplet code) which govern translation of the nucleotide sequence in mRNA into the amino acid sequence of a polypeptide Codon: a group of three consecutive nucleotides in RNA which specifies one amino acid (e.g. ‘UGG’ specifies Trp and ‘AUG’ specifies Met)

Genetic code - triplet, universal, redundant and arbitrary [Fig. 7-21] mRNA codons Corresponding amino acid that will be attached onto tRNA

mRNA can be translated in three possible reading frames

“START” and “STOP” codons on mRNA are essential for proper mRNA translation [Becker et al. The World of the Cell, Fig. 22-6]

Genetic code problem: 5’ AGUCUAGGCACUGA 3’ For the RNA sequence above, indicate the amino acids that are encoded by the three possible reading frames. If you were told that this segment of RNA was close to the3’ end of an mRNA that encoded a large protein, would you know which reading frame was used?

Frame 1-AGU CUA GGC ACU GA Frame 2-A GUC UAG GCA CUG A Frame 3-AG UCU AGG CAC UGA Frame 1-Ser – Leu – Gly - Thr

What are the main players involved in the process of translation Ribosomes [“protein synthesis machines”]: carry out the process of polypeptide synthesis tRNA [“adaptors”]: : align amino acids in the correct order along the mRNA template Aminoacyl-tRNA synthetases: attach amino acids to their appropriate tRNA molecules mRNA: encode the amino acid sequence for the polypeptide being synthesized Protein factors: facilitate several steps in the translation Amino acids: required as precursors of the polypeptide being synthesized

Ribosome Assembly Eukaryotes Large=60S Small=40S Prokaryotes Large=50S Fig. 7-28 Eukaryotes Large=60S Small=40S Prokaryotes Large=50S Small=30S

Centrifugation-separates organelles and macromolecules (panel 5-4) Heavy particles move to pellet, e.g. nucleus Fixed angle Swinging bucket

Svedberg unit, S, shows how fast a particle sediments when subject to centrifugal force. From Becker, World of the Cell, p. 327

Fig. 7-23: tRNA structure Old ‘cloverleaf’ model 3-D L-shaped model Amino acid attachment H-bonds between complementary bases anticodon

Aminoacyl tRNA synthase charge-links tRNA with amino acids [Fig 7-26] High Energy Ester bond

Fig. 7-26, part 2 tRNA anticodon binds mRNA codon

28S Fig. 7-28 5.8S Ribosomes made of rRNA and protein 18S 5S small subunit large subunit Come together during translation

Ribosomes are protein synthesis factories [Fig. 7-29]

Ribosomes can be free in the cytosol or attached to the ER membranes

Fig. 7-35: polyribosomes or polysomes

Fig. 7-32: Initiation of translation Initiator rRNA-Met +small subunit Start codon Large subunit binds

Fig. 7-32: translation Initiator tRNA in P site Aminoacyl-tRNA binds to A site Ribosome shift: one codon tRNA1 moves from P site to E site; tRNA2 moves from A site to P site Peptide bond forms

Fig. 7-30: Elongation of polypeptide Bind Bind: aminoacyl-tRNA anticodon to mRNA codon Bond Bond: make peptide bond Shift: move ribosome along mRNA, move tRNAs Shift

Stop codon at A site Fig. 7-34: termination

Fig. 7-34:termination Release factor binds stop codon at A site Release ribosome from mRNA Release C terminal from tRNA+shift

Protein destruction Proteolysis: digestion of proteins by hydrolyzing peptide bonds Proteases: enzymes that digest proteins Many proteases aggregate to form a proteasome in eukaryotes Proteins are tagged for destruction by a molecule called ubiquitin Several proteins are also degraded in lysosomes in eukaryotes

Proteasome-mediated degradation of short-lived and unwanted proteins [Fig. 7-36]

Mechanism of ubiquitin-dependent protein degradation [Becker et al. The World of the Cell, Fig. 23-38]

SUMMARY Transcription RNA processing Translation Protein destruction

Transcription of two genes seen be EM [Fig. 7-8] Where are Polymerases? Where are transcription start/stop sites? Where are the 3’ and 5’ ends of the transcript? Which direction are the RNA polymerases moving? Why are the RNA transcripts so much shorter than the length of the DNA that encodes them?