TRANSKRIPSISINTESIS RNA : mRNA, tRNA, rRNA

Dyah Ayu OktavianiePKH-UB

Study objectivesYou will need to know ALL the concepts and details in this lecture.1. What are the three main types of RNA and what are their functions?2. Understand how the following terms apply to RNA structure: phosphodiester bonds, 5' and 3 ends, nucleosides, complementary base pairing, stem loops.3. Compare and contrast DNA and RNA structure.4. What is a gene? What is gene expression? *Understand transcription, translation, and RNA processing in both prokaryotes and eukaryotes. 5. Define operons and polycistronic messages. How do they function in prokaryotic gene expression? 6. *Compare and contrast the features of prokaryotic and eukaryotic gene expression. Do eukaryotes have operons? What are exons, introns, primary transcripts, capping, tailing, and splicing. What is the 5' cap (methylguanosine cap)? How and when is the poly-A tail added to the transcript? Where does eukaryotic RNA processing occur? 7. Understand the structure and function of promoters and terminators in transcription. Contrast intrinsic terminators and rho-dependent terminators.8. Know the subunit structure of bacterial RNA polymerase and the sigma cycle.

DOGMA SENTRAL

DNA PEMBAWA INFORMASI GENETIK

• TRANSKRIPSI (SINTESIS mRNA, tRNA, rRNA)

• TRANSLASI (SINTESIS PROTEIN)

• PENGENDALIAN

TRANSKRIPSI

• Awal sintesis RNA, perpanjangan dan terminasi

• r- RNA 80%

• t- RNA 15%

• m- RNA 5%

A. Functions of the major RNAs

1. messenger RNAs (mRNA) contain genetic information to encode a protein

3. ribosomal RNAs (rRNA) are structural and catalytic component of ribosomes

2. transfer RNAs (tRNA) act as adapters between the mRNA nucleotide code and amino acids during protein synthesis

phe

2. The phosphodiester bonds of RNA are analogous to those of DNA.

3. The 5' and 3' ends of RNA are analogous to those of DNA.

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O

P-O-C5' end

ring numbering system for ribose

O

5'

2’

1’

3’

4’-C O

OP

OC

HO

O-

3’ end RNA

O

OH

OHphosphodiester

bond

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PERBEDAAN STRUKTUR DNA - RNA

KOMPOSISI BASA DNA & RNA

• DNA– A 25%– T 33– G 24– C 18

• RNA– U 25 %– A 32– C 23– G 20

Complementary base pairing

CCCUUUGGGAAA

GGGAAACCCUUU RNA

RNA

GGGAAACCCUUU RNA

CCCTTTGGGAAA DNA

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hydrogenbonding

Gene numbers

virusesprokaryotes eukaryotes

groupapproximategene number

4-200500-12,000 5,000-125,000

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Any given species has a unique setof genes that confers a unique set of properties.

Proteins and RNAs determine all of thecharacteristics of organisms and cells.

Example: Escherichia coli has 4405 genes

~117 encode RNAs (tRNA, rRNA) ~4288 encode proteins

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1 gene

1 mRNA

transcription

1 polypeptide

translation

1. Expression of single genesEx.1: a single gene that encodes a protein

Gene expression in prokaryotes

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1 genetranscription

1 RNA

degraded 1 tRNA etc.

RNA processing

Ex. 2: a single gene that encodes one rRNA or tRNA

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operontwo or more genes transcribed together

a single RNA molecule that represents more than one gene

polycistronic message

2. Expression of operons

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A B CDNA

transcription

polycistronicmRNA

1 operon

processing

rRNArRNA

degraded2 or more rRNAs

b. Operons can encode several rRNA molecules.

1 polycistronic RNA

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Important points

Most prokaryotes use operons.Operons are used to coordinategene expression and often containgenes of related function.

The details of organization, processing and degradation are different for different RNAs.

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Prokaryotic transcription

A. overviewB. transcribed regionsC. RNA polymeraseD. promotersE. terminatorsF. sigma factor

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Overview of prokaryotic transcription

RNA polymerase

primary transcript complementary to one strand of the coding region

RNA synthesis from a DNA template

typicalgene dsDNA

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Defined regions are transcribed

upstream region

transcribedregion

downstream region

promoter(RNA polymerase

binding site)

transcriptionstart site

terminationsite

gene dsDNA

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GENA

• Promoter

• Operator

• Lokus penempelan aktivator

• Struktural gena : mRNA

• Terminator

Polisistronik

Monosistronik

TRANSKRIPSI = SINTESIS RNA (mRNA, tRNA, rRNA)

– Polimerase RNA– ATP, CTP, GTP, UTP

• Urutan pada DNA:– Promoter– Operator– Situs penempelen aktivator – Terminator

Gambaran dasar sintesis RNA

• Prekursor : ATP CTP GTP UTP

• Polimerase RNA

• Reaksi polimerisasi

• Urutan RNA ditentukan DNA

• Salah satu untai DNA sebagai catakan

• Untai RNA tumbuh dari 3’

• Polimerase mulai sintesis tanpa primer

gene,or operon

RNA polymerase

C. RNA polymerase is the enzyme that synthesizes RNA from a DNA template.

complementary RNA

DNA template

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++ completed

transcriptTB

1. Subunit structure of bacterial RNA polymerase

'

core enzyme

The holoenzyme includes one of several sigma factors. TB

'

holoenzyme

Binding of polymerase to Template DNA

• Polymerase binds nonspecifically to DNA with low affinity and migrates, looking for promoter.

• Sigma subunit recognizes promoter sequence.

• RNA polymerase holoenzyme and promoter form "closed promoter complex" (DNA not unwound).

• Polymerase unwinds about 12 base pairs to form "open promoter complex“.

The sigma factor cycle

Sigma factors are needed for promoter binding, but after transcription starts they dissociate.

Sigma factors ( ) are a subunit of RNA polymerase.

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INISIASI

RNA pol holoenzyme (core + sigma)

sigmafactor

RNA (~10 nucleotides)

sigma factor

core enzyme

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+core enzyme

termination

RNA

sigma

holoenzyme TB

TAATGTGAGTTAGCTCACTCATTA

GGCACCCCAGGCTTGACATTTATG

CTTCCGGCTCGTATGTTGTGTGGA

AATTGTGAGCGGATAACAATTTCA

CACAGGAAAGAGCTATGACC...

Upstream region of the lactose operon

-35 region

-10 region (Pribnow)

Shine-dalgarno (RBS)

Translation start site

Transcription start site

PromotersSites on DNA where RNA polymerase binds to start transcription

promoter

upstream region

transcribedregion

downstream region

transcriptionstart site

terminationsite

gene dsDNA

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1. Typical bacterial 70 promoter

TTGACA TATAATAACTGT ATATTA

TATAAT = -10 consensus

sequence

TTGACA = -35 consensus

sequence

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*also called Pribnow box; ~ 10 bases before start

site of transcription

PROMOTER PROKARIOT

PROMOTER PROKARIOT & EUKARIOT

PERPANJANGAN TRANSKRIPSI

Transcriptional terminators

DNA region that mediates the termination of transcription.

gene dsDNA

region whereterminators areusually found

terminationsite

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1. Intrinsic terminatorDNA encoding an RNA that formsa stem loop followed by a run of "U"sthat is used for transcriptional termination.

UUUURNA

3' end of RNATB

The RNA stem loop binds to RNA pol and causes termination

Intrinsic terminator function

Important fact: Intrinsic terminators must betranscribed in order to function. TB

2. Rho-dependent terminator

A DNA site where RNA polymerasepauses and transcription is terminated by Rho protein

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Rho protein

Rho protein binds RNA then moves along RNA until it contacts RNA pol and terminates transcription

RNA polpauses at

Rho termination siteTB

Heksamer Rho

• Rho menempel RNA bergerak pada untai tunggal mRNA dengan menghidrolisa ATP

• Ketika Rho mencapai polimerase, memecah hibrid RNA-DNA

tRNA assists in translation

• Transfer RNA takes a specific amino acid to the ribosome

• Single stranded but has areas where bases hydrogen bond to each other– Cloverleaf shape

• 3’ end picks up the amino acid• Which amino acid is determined by 3 bases opposite the

amino acid called an anti-codon• Anti-codons pair with complementary codons in mRNA at

a ribosome

(a) Structure of 16S rRNA

(b) Structure of 23 S rRNA

STABILITAS RNA

• mRNA t ½ 2-3 menit,

• cara pengendalian