Zdroje: http://www.ncbi.nlm.nih.gov/books http://www.jblearning.com/catalog/ Lewin's GENES XI Lewin's Essential GENES, 3rd ed. http://www.garlandscience.com Molecular Biology of the Cell, 5th ed., Alberts et al. Essential Cell Biology, 4th ed., Alberts et al. http://bcs.whfreeman.com/lodish7e/ (Molecular Cell Biology, 7th ed., Lodish et al.)

http://www.biocenter.sk/ltteaching.html

Transkripcia – od DNA k proteínom •  Transkripčný aparát •  Základy génovej expresie

„Centrálna dogma“

346 CHAPTER 10 Modern Recombinant DNA Technology

its identity—information that is then used to compile the sequence of the DNA molecule. Such methods require no amplification or chemical labeling, and thereby reduce the cost and time of sequencing even fur-ther, making it possible to obtain a complete human genome sequence for under $1000 in hours.

Comparative Genome Analyses Can Identify Genes and Predict Their Function Strings of nucleotides, at first glance, reveal nothing about how that genetic information directs the development of a living organism—or even what type of organism it might encode. One way to learn something about the function of a particular nucleotide sequence is to compare it with the multitude of sequences available in public databases. Using a computer program to search for sequence similarity, one can determine whether a nucleotide sequence contains a gene and what that gene is likely to do—based on the gene’s known activity in other organisms.

Comparative analyses have revealed that the coding regions of genes from a wide variety of organisms show a large degree of sequence conser-vation (see Figure 9–19). The sequences of noncoding regions, however, tend to diverge over evolutionary time (see Figure 9–18). Thus, a search for sequence similarity can often indicate from which organism a particu-lar piece of DNA was derived, and which species are most closely related. Such information is particularly useful when the origin of a DNA sample is unknown—because it was extracted, for example, from a sample of soil or seawater or the blood of a patient with an undiagnosed infection.

But knowing where a nucleotide sequence comes from—or even what activity it might have—is only the first step toward determining what role it has in the development or physiology of the organism. The knowl-edge that a particular DNA sequence encodes a transcription regulator, for example, does not reveal when and where that protein is produced, or which genes it might regulate. To learn that, investigators must head back to the laboratory.

Analysis of mRNAs By Microarray or RNA-Seq Provides a Snapshot of Gene Expression As we discussed in Chapter 8, a cell expresses only a subset of the thou-sands of genes available in its genome. This subset differs from one cell type to another. One way to determine which genes are being expressed in a population of cells or in a tissue is to analyze which mRNAs are being produced.

The first tool that allowed investigators to analyze simultaneously the thousands of different RNAs produced by cells or tissues was the DNA microarray. Developed in the 1990s, DNA microarrays are glass micro-scope slides that contain hundreds of thousands of DNA fragments, each of which serves as a probe for the mRNA produced by a specific gene. Such microarrays allow investigators to monitor the expression of every gene in an entire genome in a single experiment. To do the analysis, mRNAs are extracted from cells or tissues and converted to cDNAs (see Figure 10–12). The cDNAs are fluorescently labeled and allowed to hybridize to the fragments on the microarray. An automated fluorescence microscope then determines which mRNAs were present in the original sample based on the array positions to which the cDNAs are bound (Figure 10–27).

Although microarrays are relatively inexpensive and easy to use, they suffer from one obvious drawback: the sequences of the mRNA samples to be analyzed must be known in advance and represented by a corre-sponding probe on the array. With the development of next-generation

WASH; SCAN FOR RED AND GREEN FLUORESCENTSIGNALS AND COMBINE IMAGES

small region of microarrayrepresenting 110 genes

mRNA from sample 1

mRNA from sample 2

ECB4 e10.33/10.28

HYBRIDIZE TO MICROARRAY

convert to cDNA,with red labeled

fluorochrome

convert to cDNA,with green labeled

fluorochrome

Figure 10–27 DNA microarrays are used to analyze the production of thousands of different mRNAs in a single experiment. In this example, mRNA is collected from two different cell samples—for example, cells treated with a hormone and untreated cells of the same type—to allow for a direct comparison of the specific genes expressed under both conditions. The mRNAs are converted to cDNAs that are labeled with a red fluorescent dye for one sample, and a green fluorescent dye for the other. The labeled samples are mixed and then allowed to hybridize to the microarray. After incubation, the array is washed and the fluorescence scanned. Only a small proportion of the microarray, representing 110 genes, is shown. Red spots indicate that the gene in sample 1 is expressed at a higher level than the corresponding gene in sample 2, and green spots indicate the opposite. Yellow spots reveal genes that are expressed at about equal levels in both cell samples. The intensity of the fluorescence provides an estimate of how much RNA is present from a gene. Dark spots indicate little or no expression of the gene whose fragment is located at that position in the array.

Analýza expresie tisícov génov: „DNA microarray“ „RNA-seq“

GÉNOVÁ EXPRESIA •  Transkripcia - vzniká jedno-

reťazcová RNA s rovnakou sekvenciou ako má kódujúci reťazec DNA.

(mRNA, rRNA, tRNA, malé RNA)

•  Translácia - mRNA je prepisovaná do sekvencií AK. tRNA a rRNA sú zložkami proteosyntetického aparátu. Len kódujúca časť mRNA je prepisovaná.

Gény môžu byť exprimované s odlišnou účinnosťou

Možná amplifikácia v každom z tých 2 stupňov umožňuje rozdielne hladiny rôznych proteínov

Rôzne hladiny regulácie génovej expresie

Chemická štruktúra RNA

Jazyk je takmer rovnaký - cukor je (deoxy) ribóza. Uracil je namiesto tymínu.

RNA má odlišnú štruktúru !!

RNA nesie informáciu, ale tiež môže vykonávať iné funkcie ako napríklad regulácie (iRNA), post- transkripčné modifikácie RNA, alebo katalýzu (ribozýmy).

V čom sa transkripcia odlišuje od replikácie ?

ssRNA je po syntéze vytesnená z dsDNA – tá sa po transkripcii okamžite reformuje na dvoj-závitnicovú molekulu

Väčšina RNA molekúl je oveľa kratších ako DNA – len niekoľko tisíc nukleotidov dlhé

(U eukaryotov každá mRNA nesie informáciu len pre 1 gén; u prokaryotov to môže byť aj niekoľko génov z jedného operónu

Okienko RNA-DNA helixu sa pohybuje pozdĺž DNA spolu s RNA polymerázou

Súčasne je syntetizovaných veľa RNA molekúl

Syntéza rRNA – sú viditeľné aj ribozomálne proteíny, ktoré sa v priebehu transkripcie pripájajú k rRNA (asemblácia ribozómov).

Niektoré odlišnosti medzi RNA polymerázou a DNA polymerázou:

- spája ribonukleotidy

- odštartuje reťazec RNA bez primeru

- transkripcia nie je tak precízna – 1 chyba každých 104 nukleotidov versus 1 každých 107 v DNA

Prečo je to OK?

Niektoré odlišnosti medzi RNA polymerázou a DNA polymerázou:

-- spája ribonukleotidy

-- odštartuje reťazec RNA bez primeru

-- transkripcia nie je tak precízna – 1 chyba každých 104 nukleotidov versus 1 každých 107 v DNA

Prečo je to OK? - RNA neslúži na stále uskladnenie informácie

Rôzne druhy a funkcie bunkových RNA

Rôzne druhy a funkcie bunkových RNA

MikroRNA (21 – 24 nukl.) regulujú génovú expresiu.

Rôzne druhy a funkcie bunkových RNA

micF je príkladom antisense RNA

Štruktúra a funkcie tRNA

•  Aminoacyl-tRNA - kovalentná väzba medzi COOH skupinou AK a -OH skupinou (3’- alebo 2’-) poslednej bázy tRNA.

•  AA-tRNA syntáza - katalyzuje tvorbu kovalentnej väzby.

•  Antikodón v tRNA je komplementárny ku kodónu v mRNA.

stonka

sľučka

L-podobná štruktúra maximálne oddeľuje antikódon od AK

•  74 až 95 báz

tRNA1Tyr

tRNA2Tyr

Ala-tRNA

Párovanie G-C, A-U ale aj G.U resp. G.Ψ.

mRNA je prepisovaná ribozómami nascentný reťazec

polyzómy 70S E. coli (50S + 30S) a 80S cicavce (60S + 40S).

30-35 AK rastúceho reťazca je chránených v ribozóme

Koľko rôznych mRNA a tRNA obsahuje E. coli?

mRNA je transkribovaná, translatovaná a degradovaná v baktériách súčasne

mRNA baktérie je nestabilná polycistronická mRNA má rôzne segmenty

Rýchlosť transkripcie 40 nukl./s = 15 AK/s.

Životný cyklus eukaryotickej mRNA je podstatne dlhší

Konce mRNA sú modifikované pridaním ďalších nukleotidov

Polčas života mRNA: •  S. cerevisiae - 1 - 60 min. •  vyššie organizmy - 1 - 24 hod.

Počiatočná sekvencia transkriptu je: 5’pppA/GpNpNpNp... Gppp + 5’pppApNpNp.. (guanylyltransferáza) 5’-5’ GpppApNpNp.. + pp + p („cap“) (guanine-7-metyltransferáza) cap 0 (2’-O-metyltransferáza) cap 1, 2.

K nukleázam citlivý 5’-koniec mRNA musí mať čiapočku

- “CAP” blokuje 5’- koniec mRNA a môže byť metylovaný v určitých polohách

3’- koniec mRNA je polyadenylovaný

•  polyA nie je kódovaná v DNA, •  polyA je asociovanou s PABP - 1

PABP (70kD) na 10-20 báz A. •  dodáva stabilitu mRNA •  využíva sa pri purifikácii mRNA

a následnej syntéze cDNA.

cDNA je jednovláknová DNA, ktorá je komplementárna k určitej RNA a je syntetizovaná pomocou reverznej transkriptázy in vitro.