Tran Skrip Tion

7/27/2019 Tran Skrip Tion

1/35

Chapter 5: Gene Expression - Transcription

When a protein is needed by a cell, the genetic code for that protein

must must be read from the DNA and processed.

A two step process:

1. Transcription = synthesis of a single-stranded RNA moleculeusing the DNA template (1 strand of DNA is transcribed).

2. Translation = conversion of a messenger RNA sequence into theamino acid sequence of a polypeptide (i.e., protein synthesis)

Both processes occur throughout the cell cycle.


2/35

Four different types of RNA, each encoded by different genes:

1. mRNA Messenger RNA, encodes the amino acid sequenceof a polypeptide

2. tRNA Transfer RNA, transports amino acids to ribosomesduring translation

3. rRNA Ribosomal RNA, forms complexes called ribosomeswith protein, the structure on which mRNA istranslated

4. snRNA Small nuclear RNA, forms complexes with proteinsused in eukaryotic RNA processing (e.g., exonsplicing and intron removal).


3/35

Transcription: How is an RNA strand synthesized?

1. Regulated by gene regulatory elements within each gene.

2. DNA unwinds next to a gene.

3. RNA is transcribed 5 to 3 from the template (3 to 5).

4. Similar to DNA synthesis, except:

NTPs instead of dNTPs (no deoxy-)

No primer

No proofreading

Adds Uracil (U) instead of thymine (T)

RNA polymerase


4/35

Fig. 5.2


5/35

Three Steps to Transcription:

1. Initiation

2. Elongation

3. Termination

Occur in both prokaryotes and eukaryotes.

Elongation is conserved in prokaryotes and eukaryotes.

Initiation and termination proceed differently.


6/35

Step 1-Initiation, E. colimodel:

Fig. 5.3

Each gene has three regions:

1. 5 Promoter, attracts RNA polymerase

-10 bp 5-TATAAT-3-35 bp 5-TTGACA-3

2. Transcribed sequence, or RNA coding sequence

3. 3 Terminator, signals the stop point


7/35

Step 1-Initiation

1. RNA polymerase combines with sigma factor (a polypeptide) tocreate RNA polymerase holoenzyme

Recognizes promoters and initiates transcription.

Sigma factor required for efficient binding and transcription.

Different sigma factors recognize different promotersequences.

2. RNA polymerase holoenzyme binds promoters and untwists DNA

Binds loosely to the -35 promoter (DNA is d.s.)

Binds tightly to the -10 promoter and untwists

3. Different types and levels of sigma factors influence the level anddynamics of gene expression (how much and efficiency).


8/35

Fig. 5.4


9/35

Step 2-Elongation

1. After 8-9 bp of RNA synthesis occurs, sigma factor is released andrecycled for other reactions.

2. RNA polymerase completes the transcription at 30-50 bp/second.

3. DNA untwists rapidly, and re-anneals behind the enzyme.

4. Part of the new RNA strand is hybrid DNA-RNA, but most RNA isdisplaced as the helix reforms.


10/35

Fig. 5.4


11/35

Step 3-Termination

Two types of terminator sequences occur in prokaryotes:

1. Type I (-independent)Palindromic, inverse repeat forms a hairpin loop and is believedto physically destabilize the DNA-RNA hybrid.

2. Type II (-dependent)

Involves factor proteins, believed to break the hydrogen bondsbetween the template DNA and RNA.

Fig. 5.5


12/35

Prokaryotes possess only one type of RNA polymerase

transcribes mRNAs, tRNAs, and rRNAs

Transcription is more complicated in eukaryotes

Eukaryotes possess three RNA polymerases:

1. RNA polymerase I, transcribes three major rRNAs 12S, 18S, 5.8S

2. RNA polymerase II, transcribes mRNAs and some snRNAs

3. RNA polymerase III, transcribes tRNAs, 5S rRNA, and snRNAs

*S values of rRNAs refer to molecular size, as determined in a sucrosegradient (review box 5.1)


13/35Peter J. Russell, iGenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.

Box Fig. 5.1 Sucrose density gradient centrifugation technique for separating andisolating RNA molecules in a mixture


14/35

Transcription of protein-coding genes by RNA polymerase II

Recall that RNA polymerase II transcribes mRNA

Like prokaryotes, eukaryotes require a promoter, two types

1. Basal elements (near the transcription start, ~-25 bp)

TATA Box = TATAAAA

*AT-rich DNA is easier to denature than GC-rich DNA

2. Proximal elements (located upstream, ~-50 to -200 bp)

Cat Box = CAAT and GC Box GGGCGG

Different combinations occur near different genes


15/35

Transcription of protein-coding genes by RNA polymerase II

Transcription factors (TFs) also are required by RNA polymerases(function is similar to sigma factor).

TFs are proteins, assembled on basal promoter elements

Each TF works with only one kind of RNA polymerase (required byall 3 RNA polymerases).

Numbered (i.e., named) to match their RNA polymerase.

TFIID, TFIIB, TFIIF, TFIIE, TFIIH

Binding of TFs and RNA polymerase occurs in a set order inprotein coding genes.

Complete complex (RNA polymerase + TFs) is called a pre-initiation complex (PIC).


16/35

Fig. 5.6, Order of binding is: IID + IIB + RNA poly. II + IIF +IIE +IIH


17/35


18/35

Production of the mRNA molecule (Fig. 5.7):

Three main parts:

1. 5 untranslated region (UTR) leader sequence

2. Coding sequence, specifies amino acids to be translated

3. 3 untranslated region (UTR) trailer sequence


19/35

mRNA Differences between prokaryotes and eukaryotes:

Prokaryotes

1. mRNA transcript is mature, and used directly for translationwithout modification.

2. Since prokaryotes lack a nucleus, mRNA also is translated onribosomes before is is transcribed completely (i.e., transcriptionand translation are coupled).

3. Prokaryote mRNAs are polycistronic, they contain amino acidcoding information for more than one gene.

Eukaryotes

1. mRNA transcript is not mature (pre-mRNA); must be processed.

2. Transcription and translation are not coupled (mRNA must firstbe exported to the cytoplasm before translation occurs).

3. Eukaryote mRNAs are monocistronic, they contain amino acidsequences for just one gene.


20/35

Fig. 5.8, Prokaryotes and Eukaryotes


21/35

Production of mature mRNA in eukaryotes:

1. 5 cap

After 20-30 nucleotides have been synthesized, the 5-end ofthe mRNA is capped 5 to 5 with a guanine nucleotide.

Results in the addition of two methyl (CH3) groups.

Essential for the ribosome to bind to the 5 end of the mRNA.

2. Poly (A) tail,

50-250 adenine nucleotides are added to 3 end of mRNA.

Complex enzymatic reaction (illustrated in Fig. 5.10).

Stabilizes the mRNA, and plays an important role intranscription termination.

3. Introns (non-coding sequences between exons) are removed andexons (amino acid coding sequences) are spliced.


22/35

Introns and exons:

Eukaryote pre-mRNAs often have intervening introns that must beremoved during RNA processing (as do some viruses).

intron = non-coding DNA sequences between exons in a gene.

exon = expressed DNA sequences in a gene, code for amino acids.

1993: Richard Roberts (New England Biolabs) & Phillip Sharp (MIT)

Fig. 5.11


23/35

mRNA splicing of exons and removal of introns:

1. Introns typically begin with a 5-GT(U) and end with AG-3.

2. Cleavage occurs first at the 5 end of intron 1 (between 2 exons).

3. The now free G joins with an A at a specific branch point sequencein the middle of the intron, using a 2 to 5 phosphodiester bond.

Intron forms a lariat-shaped structure.

4. Lariat is excised, and the exons are joined to form a spliced mRNA.

5. Splicing is mediated by splicosomes, complexes of small nuclearRNAs (snRNAs) and proteins, that cleave the intron at the 3 endand join the exons.

6. Introns are degraded by the cell.


24/35

Fig. 5.11


25/35

Fig. 5.13


26/35

e.g., Fig. 5.13


27/35

Post-transcriptional modification, mRNA editing:

1. Adds or deletes nucleotides from a pre-RNA, or chemically altersthe bases, so the mRNA bases do not match the DNA sequence.

2. Can results in the substitution, addition, or deletion of amino acids(relative to the DNA template).

3. Generally cell or tissue specific.

4. Examples; protozoa, slime molds, plant organelles, mammals


28/35

Genes that do not code proteins also are transcribed:

1. rRNA, ribosomal RNA

Catalyze protein synthesis by facilitating the binding of tRNA(and their amino acids) to mRNA.

2. tRNA, transfer RNA

Transport amino acids to mRNA for translation.

3. snRNA, small nuclear RNA

Combine with proteins to form complexes used in RNAprocessing (splicosomes used for intron removal).


29/35

1. Synthesis of ribosomal RNA and ribosomes:

1. Cells contain thousands of ribosomes.

2. Consist of two subunits (large and small) in prokaryotes andeukaryotes, in combination with ribosomal proteins.

3. E. coli 70S model:

50S subunit = 23S (2,904 nt) + 5S (120 nt) + 34 proteins

30S subunit = 16S (1,542 nt) + 20 proteins

4. Mammalian 80S model:

60S subunit = 28S (4,700 nt) +5.8S (156 nt) + 5S (120 nt) +50 proteins

40S subunit = 18S (1,900 nt) + 35 proteins

5. DNA regions that code for rRNA are called ribosomal DNA (rDNA).

6. Eukaryotes have many copies of rRNA genes tandemly repeated.


30/35

1. Synthesis of ribosomal RNA and ribosomes:(continued):

7. Transcription occurs by the same mechanism as protein-codinggenes, but generally using RNA polymerase I.

8. rRNA synthesis requires its own array transcription factors (TFs)

9. Coding sequences for RNA subunits within rDNA genes containinternal (ITS), external (ETS), and nontranscribed spacers (NTS).

10. ITS units separate the RNA subunits through the pre-rRNA stage,

whereupon ITS & ETS are cleaved out and rRNAs are assembled.

11. Subunits of mature ribosomes are bonded together by H-bonds.

12. Finally, transported to the cytoplasm to initiate protein synthesis.


31/35

Fig. 5.16, Mammalian example of 80S rRNA


32/35

Fig. 5.18

2 S th i f tRNA


33/35

2. Synthesis of tRNA:

1. tRNA genes also occur in repeated copies throughout the genome,and may contain introns.

2. Each tRNA (75-90 nt in length) has a different sequence thatbinds a different amino acid.

3. Many tRNAs undergo extensive post-transcription modification,especially those in the mitochondria and chloroplast.

4. tRNAs form clover-leaf structures, with complementary base-

pairing between regions to form four stems and loops.

5. Loop #2 contains the anti-codon, which recognizesmRNA codons during translation.

6. Same general mechanism using RNA polymerase III, promoters,

unique TFs, plus posttranscriptional modification from pre-tRNA.


34/35


35/35

3. Synthesis of snRNA (small nuclear RNA):

Form complexes with proteins used in eukaryotic RNA processing,splicing of mRNA after introns are removed.

Transcribed using RNA polymerase II or III.

Documents

Tran Skrip Tion