Transcription in Prokaryotes

by Jean-Pierre Herveg, Etienne De Plaen and a lot of friends atthe Brussels Branch of the Ludwig Institute for Cancer research (Licr) and the Christian de Duve*

Institute for cellular Patholgy (ICP).

April 2006Université Catholique de Louvain

Avenue E. Mounier, 1200 Brussels (Belgium)

Questions

1 what is the meaning of the sentence :” The sequence of a prokaryotic proteinis colinear with the translated mRNA

2. which sugar replaces desoxyribose in ARN ?3. which base anneals to uracil ?4. what’s a polysome ?5. what’s an operon, an operator ? Describe !6. what is X-gal.7. what is lacZ? 8. what is IPTG?9. describe a promoter.10. what is the role of sigma in prokaryotes11. why are there a pUR278, pUR289 and a PUR288 to make fusion proteins12. explain the control of protein production by pET-3a

Transcription in prokaryotes

The sequence of a prokaryotic protein is colinear with the translated mRNA;that is, the transcript of the gene is the molecule that is translated into the polypeptide.In prokaryotes, ther is no nucleus and then replication, transcription and translation take place inThe same compartment.

Reverse transcription is the oppositeThe enzymes involved are RNA polymerase for transcription and reverse transcriptase for the opposite.

--------------------------questionwhat is the meaning of the sentence :” The sequence of a prokaryotic protein is colinear withthe translated mRNA

Ribose:Is a sugar containing 5 carbon atoms and a hydroxyle in the position 2’UracileIs a RNA base which is represented by a U. U anneals with A.

--------------------------------------La ribosaEs un azúcar de 5 carbonos. Es un componente estructural de la estructura del ARN, como el ATP, GTP, CTP y TTP.UraciloEs una de las 4 bases del ARN. Se representa con la letra U. En el ARN, El uracilo reemplaza a la timina.El uracilo se aparea con la adenina.---------------------------------------Cuestiones para el examen1. ¿ Cuál es el azúcar y la basa específicos del ARN2. ¿ Con cuál base se aparea el uralcilo?

There are 5 types of RNA, each encoded by its own type of gene:

mRNA -(80 % in eucaryoyes) Messenger RNA: Encodes the amino acid sequence of a polypeptide.

tRNA - (15 % in eucaryoyes) Transfer RNA: Brings amino acids to ribosomes during translation.

mRNA - (5 % in eucaryoyes) Ribosomal RNA: with ribosomal proteins, makes up the ribosomes.rRNA possess an enzymic activity. The ribosome is the organelles that translate the mRNA into polypeptide.

snRNA - Small nuclear RNA: With proteins, forms complexes that are used in RNA processing

siRNA and miRNA: are inhibitor od gene expression

Transcription and translation take place in the same compartment.

In prokaryotes, there is simultaneous transcription and translation.

Here, in E. Coli you can see a long fiber running from top to bottom (green arrow ). This fiber is aSegment of the E. coli chromosome (dsDNA).

This picture comes from:http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/M/Miller_Hamkalo.html

Extending from the DNA are polysomes (red arrow), A polysome is a backbone of messenger RNA (mRNA) to which ribosomes are attached. On the left picture, you can see that the size of polysomes increases from top to bottom.

Each polysome is attached to the DNA fiber by a complex of proteinsthat includes a molecule of RNA polymerase.

Thus

the DNA is transcribed by RNA polymerase molecules moving from top to bottom, and

the growing mRNA molecules are translated by ribosomesmoving in a proximal -> distal direction.

In E. coli, then, and probably in all prokaryotes,the transcription of DNA into mRNA and the translation of mRNA intopolypeptides (not visible here) are closely coordinatedin both time and space.

(In eukaryotes, in contrast, while all transcription takes place in thenucleus, most (but not all) translation of mRNA occurs later in the cytosol).

Defintion:

An operon is a group of genes including a common promoter and an operator, which control the transcription of these genes to produce a common messenger RNA (mRNA).

A promoter is a DNA sequence that enables a gene to be transcribed.The promoter is recognized by RNA polymerase, which then initiates transcription.

An operator is a segment of DNA that recognized by a regulatory protein (inducer or or repressor).

In negative inducible operons (the lac operon), a regulatory repressor protein is normallybound to the operator and it prevents the transcription of the genes on the operonIf an inducer molecule is present, it binds to repressor and changes its conformationso that it is unable to bind to the operator.This allows for the transcription of the genes controlled by the operator.

In negative repressible operons, (the trp operon) transcription of the genes on the operonnormally takes place. Repressor proteins are produced by a regulator gene but they areunable to bind to the operator in their normal conformation. However certain moleculescalled corepressors can bind to the repressor protein and change its conformation so thatit can bind to the operator. The activated repressor protein binds to the operator andprevents transcription.

Operons can also be positively controlled. With positive control, an activator proteinstimulates transcription by binding to DNA (usually at a site other than the operator).

a negative inducible operons:the lac operon consists of a regulatory gene and 3 structural genes (z, y, and a).The i gene codes for a repressor. The z gene codes for ß-galactosidase, which hydrolyses lactose intogalactose and glucose. The y gene codes for permease, which increases permeability to ß-galactosides.The a gene encodes a transacetylase.

In the presence of an inducer of the lac operon, the repressor protein binds the inducer. RNA polymerase isthus able to bind at the promoter region, and transcription of the operon ensues.

Lactose analogues

1. IPTG is an inducer (acts like lactose, but is not metabolized byß-galactosidase).

A number of lactose derivatives or analogs have been described that are useful for work with the lac operon.These compounds are mainly substituted galactosides, where the glucose moiety of lactose is replacedby another chemical group.

Isopropyl-β-D-thio-galactoside (IPTG) is frequently used as an inducer of the lac operonfor physiological work. IPTG binds to repressor and inactivates it, but is not a substrate for β-galactosidase.One advantage of IPTG for in vivo studies is that it cannot be metabolized by E. coli, therefore the growthrate of cells (usually maintained with glycerol as the carbon and energy source), is not a variablein the experiment.

In addition, IPTG is transported efficiently independent of whether the lacY geneis functional. And since cells don't metabolize IPTG, its concentration doesn't change during the courseof an experiment.

Lactose analogues

1. X-gal (is metabolizide and gives a blue reaction product).

X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactoside) turns colonies which produceβ-galactosidase blue. IPTG is used in what is called the white andblue screening.

http://en.wikipedia.org/wiki/Lac_operon

a negative repressible operon:

The trp operon encodes the genes for the synthesis of tryptophan.This cluster of genes is regulated by a repressor that could bind to the operator sequence.The activity of the trp repressor for binding the operator region is enhanced when it binds tryptophan: tryptophan is known as a corepressor.

Since the activity of the trp repressor is enhanced in the presence of tryptophanthe rate of expression of the trp operon is graded in response to the level of tryptophan in the cell (attenuationElement).

---------------------------------Cuestiones para el examen1. Definir la palabra opéron en las bacterias2. ¿ Que es que X-gal.3. ¿ Que es que lacZ? 4. ¿ Que es que IPTG?5. describir el opéron lac, trp…

----------------------------------------------to stall: calar (autocalar)

Fine regulation of the tryptophane operon.

Expression of the trp operon is also regulated by attenuation (attenuator element).The attenuator region is involved in controlling transcription from the operon after RNA polymerasehas initiated synthesis.

Beginning of transcription

The RNA pol is a tetrameric enzyme. The factor sigma helps to recognize the promoters.

The genetic information contained in DNA is transcribed to RNA by a transcription complexIncluding DNA-directed RNA polymerase (RNA Pol). A bacterial transcription initiation complexcomprised of the core RNA Pol enzyme and a (sigma) factor binds to a promoter and,upon initiation of RNA synthesis, releases the factor.http://dnaresearch.oxfordjournals.org/cgi/content/short/dsi016v1

Institution: Bolivia: PNAS SponsoredPublished online before print March 27, 2006,PNAS | April 4, 2006 | vol. 103 | no. 14 | 5332-5337

Biological Science / BiochemistryInsights into transcriptional regulation and {sigma} competition from anequilibrium model of RNApolymerase binding to DNAIrina L. Grigorova, Naum J. Phleger, Vivek K. Mutalik, and Carol A. Gros

In bacteria transcription is initiated by RNA polymerase (RNAP) holoenzyme (E which is formed

when core RNAP (E) binds the transcription initiation factor (1). Einitially binds to promoter sitesin a closed complex, which then transits to an open complex, competent for transcription.The number of intermediates between the closed and open complex is variable and promoter-dependent;each step may be subject to regulation in vivo (2, 3). At least for some promoters, Ebinding to promotersis thought to be reversible on the time scale of transcription initiation in vivo (3); reversibility hasalso been demonstrated in vitro for several promoters (3–6). Even binding to the strong lac UV5promoter is reversible in vitro when tested under conditions that approximate the in vivo situation (6).

Recruitment of E to promoters in vivo is thought to depend on the intrinsic binding affinity of thepromoter and is modulated by repressors that prevent and activators that stabilize interactions betweenE and the promoter (3). Based on in vitro studies of the mechanism of activator function, it is believed

that promoters that bind E weakly require activators to recruit E In addition, cells contain multiple ,

which direct E to various sets of promoters specific to the factors (1). These s are believed to competewith each other for binding to E (7–10). By changing the relative levels of the {sigma}s, Escherichia coli isthought to coordinate its transcriptional program with growth conditions (11–13). This view is basedon observations indicating that (i) overexpressing one decreases expression of genes controlled

by another (7), (ii) mutationally altering binding constants of one for E, alters expression by another (14),

and (iii) physiological effectors such as ppGpp may act by altering relative binding of s to E (8–10).In the present work, we use an equilibrium model of RNAP binding to DNA to explore in vivo scenariosthat permit transcription regulation by activator recruitment of RNAP and E competition.

The promoter is recognized by RNA polymerase and the factor. RNA pol then

initiates transcription. When the transcript is around 10 nt long, the factorquits the complex

------------------------------Los promotores bacterianos poseen cuatro características comunes:(1) lel punto de comienzo de la transcripción, denominado +1,(2) la secuencia de la posición –10 llamada caja de Pribnow (TATAAT),(3) la secuencia –35 (TGTTGACA) y (4) la distancia entre las secuencias –10 y –35. E punto donde se inicia la transcripción es

genralmenteuna purina (A ou G).

the end of transcription: terminationTwo termination mechanisms are well known:

Rho-independent termination involves terminator sequences within theRNA that signal the RNA polymerase to stop. The terminator sequence is usually a palindromic sequencethat forms a stem-loop hairpin structure that leads to the dissociation of the RNA Pol from the DNA template.

Rho-dependent termination uses a termination factor called (rho) factor to stop RNA synthesis at specific sites.This protein binds and runs along the mRNA towards the RNA Pol. When the (rho) factor reaches the RNA Polit causes RNA Pol to dissociate from the DNA, terminating transcription.

Other termination mechanisms include where RNA Pol comes across a region with repetitious thymidineresidues in the DNA template. or where a GC-rich inverted repeat followed by 4 A residuesthe inverted repeat forms a stable stem loop structure in the Rna, which causes the RNA to dissociate fromthe DNA template.

Translation (genetic code)

codon iniciador AUGStop: TAA, TAG, TGA

Acido Aspartico : Asp = DAcido glutamico : Glu = EAlanina : Ala = AArginina : Arg = RAsparagina : Asp = NCisteina : Cys = Cfenilalana : Phe = FGlicina : Gly = GGlutamina : Gln = QHistidina : His = Hisoleucina :Ile = ILeucina :Leu = LLisina : Lys = KMetioniona : Met = MProlina : Pro = PSerina : Ser = STirosina : Tyr = YTreonina : Thr = TTriptofana : Trp = WValina : Val = V

Table of the codons and amino acids(one letter code)

uuu F ucu S uau Y ugu Cuuc F ucc S uac Y ugc Cuua L uca S uaa stop tga stopuug L ucg S uag stop ugg W

cuu L ccu P cau H cgu Rcuc L ccc P cac H cgc Rcua L cca P caa Q cga Rcug L ccg P cag Q cgg R

auu I acu T aau N agu Sauc I acc T aac N agc Raua I aca T aaa K aga Ratg M acg T aag K agg R

guu V gcu A gau D ggu Gguc V gcc A gac D ggc Ggua V gca A gaa E gga Ggug V gcg A gag E ggg G

Free tRNAs are loaded with their specific amino-acyl by anAminoacyl-tRNA transferase.They are now ready to participate to translation.Free tRNA, are made of a short RNA sequence, in which they are anticodons

tRNA are made of a short RNA sequence, in which they are anticodons they canBe loaded with an aùino-acyl.In the ribosome (large subunit), two sites A and P can be loaded each withaminoacyl-tRNA.A peptide bond is the made between thes two aminoacyls.

Then, the tRNA molecule is expelled see the black arrow below.It reached the place where it could be loaded with another aminoacyl.The ribosome moves (scarlet arrows, the tRNA in the second site moves in the first oneAnd another aminoacyl-tRNA takes its place.

Comparaison between prokaryotes and eukaryotes:

fused proteins

pUR278

pMAL

non fused proteins

pET-3a

----------------------------questionexplain the control of protein production by pET-3a

pQE (Quiagen), HIS tag

Exercises: recombinant insulin and growth hormone

Search for the insulin gene:Database: Gene (1)For: INS or INSULIN, both are okay.The click on Go

You should first read the Summary…. And remember the significance of signal peptide, post Translationnaly, disulfide bonds, glucose receptor and glucose uptake.What’s a minus strand ?Does this gene have an intron ?On what chromosome stand the gene ?What is the meaning of p in 11p5.5 ?

Now, click on NM_000207.1 (DEFINITION homo sapiens insulin (INS), mRNA.

recombinant human insulin rh-insulinLOCUS NM_000207 450 bp mRNA linear PRI 29-JAN-2006DEFINITION Homo sapiens insulin (INS), mRNA.

1 gctgcatcag aagaggccat caagcacatc actgtccttc tgccatggcc ctgtggatgc 61 gcctcctgcc cctgctggcg ctgctggccc tctggggacc tgacccagcc gcagcctttg 121 tgaaccaaca cctgtgcggc tcacacctgg tggaagctct ctacctagtg tgcggggaac 181 gaggcttctt ctacacaccc aagacccgcc gggaggcaga ggacctgcag gtggggcagg 241 tggagctggg cgggggccct ggtgcaggca gcctgcagcc cttggccctg gaggggtccc 301 tgcagaagcg tggcattgtg gaacaatgct gtaccagcat ctgctccctc taccagctgg 361 agaactactg caactagacg cagcccgcag gcagcccccc acccgccgcc tcctgcaccg 421 agagagatgg aataaagccc ttgaaccagc /translation="MALWMRLLPLLALLALWGPDPAAAFVNQHLCGSHLVEALYLVCERGFFYTPKTRREAEDLQVGQVELGGGPGAGSLQPLALEGSLQKRGIVEQCCTSICSLYQLENYCN" proprotein 45..374 (329 = 100%)

sig_peptide 45..116 (71/329= 0.215) atggcc ctgtggatgc gcctcctgcc cctgctggcg ctgctggccc tctggggacc tgacccagcc gcagcc mat_peptide 117..374 ( /product="proinsulin" mat_peptide 117..206 (89/329= 0,27)tttgtgaaccaaca cctgtgcggc tcacacctgg tggaagctct ctacctagtg tgcggggaac gaggcttctt ctacacaccc aagac /product="proinsulin peptide B" mat_peptide 312..374 (62/329= 0.188)ggcattgtg gaacaatgct gtaccagcat ctgctccctc taccagctgg agaactactg caac

/product="proinsulin peptide A”In between 0.327 %

Summary: After removal of the precursor signal peptide, proinsulin is post-translationally cleaved into two chains(peptide A and peptide B) that are covalently linked via two disulfide bonds.Binding of this mature form of insulin to the insulin receptor (INSR) stimulates glucose uptake.

Official Symbol: INS and Name: insulin [Homo sapiens] Other Designations: proinsulin Chromosome: 11; Location: 11p15.5 GeneID: 3630

The mRNA is translated in a protein which have four domains: The hydrophobic siganl peptide is cleaved in the ERf the eukaryotic cell. The C peptide is cleaved in the blood by proteases.

At the end of this process, we are left with this structure:

The cDNA coding proinsulin is used to make protein fusion with B-gal. An atg is added at the beginnning.The fusion protein is purified. The ß-gal beginning ic cleaved by cyanogen bromide. The c peptide is cleaved by A protease.

atg + tttg 121 tgaaccaaca cctgtgcggc tcacacctgg tggaagctct ctacctagtg tgcggggaac 181 gaggcttctt ctacacaccc aagacccgcc gggaggcaga ggacctgcag gtggggcagg 241 tggagctggg cgggggccct ggtgcaggca gcctgcagcc cttggccctg gaggggtccc 301 tgcagaagcg tggcattgtg gaacaatgct gtaccagcat ctgctccctc taccagctgg 361 agaactactg caactag

human growth hormone (hGH)

Official Symbol: GH1 and Name: growth hormone 1 [Homo sapiens] Other Aliases: GH, GH-N, GHN, hGH-N Other Designations: pituitary growth hormone Chromosome: 17; Location: 17q24.2 GeneID: 2688

The protein encoded by this gene is a member of the somatotropin/prolactin family of hormoneswhich play an important role in growth control. The gene, along with four other related genes,is located at the growth hormone locus on chromosome 17 where they are interspersed in thesame transcriptional orientation; an arrangement which is thought to have evolved by a seriesof gene duplications. The five genes share a remarkably high degree of sequence identity.Alternative splicing generates additional isoforms of each of the five growth hormones,leading to further diversity and potential for specialization. This particular family member is expressedin the pituitary but not in placental tissue as is the case for the other four genes in the growth hormonelocus. Mutations in or deletions of the gene lead to growth hormone deficiency and short stature.

LOCUS NM_022562 376 bp mRNA linear PRI 26-FEB-2006DEFINITION Homo sapiens growth hormone 1 (GH1), transcript variant 5, mRNA.ACCESSION NM_022562VERSION NM_022562.2 GI:20809252

1 aggatcccaa ggcccaactc cccgaaccac tcagggtcct gtggacagct cacctagctg 61 caatggctac agaggctgga agatggcagc ccccggactg ggcagatctt caagcagacc 121 tacagcaagt tcgacacaaa ctcacacaac gatgacgcac tactcaagaa ctacgggctg 181 ctctactgct tcaggaagga catggacaag gtcgagacat tcctgcgcat cgtgcagtgc 241 cgctctgtgg agggcagctg tggcttctag ctgcccgggt ggcatccctg tgacccctcc 301 ccagtgcctc tcctggccct ggaagttgcc actccagtgc ccaccagcct tgtcctaata 361 aaattaagtt gcatca

FEATURES Location/Qualifiers source 1..376 /organism="Homo sapiens" gene 1..376 /gene="GH1" /note="synonyms: GH, GHN, GH-N, hGH-N" CDS 63..155 /gene="GH1"

/translation="MATEAGRWQPPDWADLQADLQQVRHKLTQR" misc_feature 72^73 /gene="GH1" /note="Region: location of alternate exons 2, 3 and 4" STS 197..336 (STS= sequence tagged sites)

see http://cfern.bio.utk.edu/journal/Hamilton1998/THESIS_A1.htm polyA_signal 357..362 polyA_site 376

atggcta agaggctgga agatggcagc ccccggactg ggcagatctt caagcagacctacagcaagt tcgacacaaa ctcacacaac gatga