(CHAPTER 12- Brooker Text)

Transcription

Sept 18 & 23, 2008BIO 184

Dr. Tom Peavy

• Bacterial mRNA may be polycistronic, which means it encodes two or more polypeptides

• Transcription occurs in three stages– Initiation– Elongation– Termination

• These steps involve protein-DNA interactions – Proteins such as RNA polymerase interact with DNA

sequences

The Stages of Transcription

• promoter = recognition site for transcription factors

• transcription factors + RNA polymerase to bind to the promoter

(initial phase) = closed promoter complex

• then DNA is denatured (bubble) = open promoter complex

Initiation

• E. coli RNA polymerase = holoenzyme – Core enzyme (Four subunits = 2’)

– Sigma factor (One subunit =

Initiation stages involve RNA pol holoenzyme• Binding loosely to the DNA• Scaning for promoter region• Forming Open promoter complex• Synthesizing Short stretch of RNA• Releasing Sigma factor

Prokaryotic Transcription

Sequence elements that play a key role in

transcription

(pribnow box)

Bacterial Promoter

• The RNA transcript is synthesized during ELONGATION step

• The DNA strand used as a template for RNA synthesis is termed the template or noncoding strand

• The opposite DNA strand is called the coding strand– It has the same base sequence as the RNA transcript

• Except that T in DNA corresponds to U in RNA

• short RNA-DNA hybrid is forced to separate

= release of newly made RNA

• E. coli has two different mechanisms for termination– 1. rho-dependent termination

• Requires a protein known as (rho)

– 2. rho-independent termination• Does not require but uses stem loop structure and U–rich

sequence region

Termination of Bacterial Transcription

rho utilization site

-dependent terminationFigure 12.8

Rho protein is a helicase

-dependent terminationFigure 12.8

Types of RNA

mRNA = messenger RNA encodes for the sequence of amino acids within a polypeptide

tRNA = transfer RNA carries amino acids at the amino acyl terminus for incorporation into growing polpeptides during translation at ribosome

rRNA = ribosomal RNA is a major component of the ribosomal complex essential for coordinating protein sythesis using the mRNA as a template (translation)

• Similar but more complex– Larger organisms– Cellular complexity– Multicellularity

TRANSCRIPTION IN EUKARYOTES

• Three Different RNA polymerase needed

1) RNA pol I = rRNA genes (except 5S)

2) RNA pol II = mRNAs (structural genes)

3) RNA pol III = tRNAs and 5S rRNA

• The core promoter is relatively short = TATA box

- determines start point for transcription

- basal transcription by itself (low level)

• Regulatory Elements (e.g. GC and CAAT boxes)

- Enhancers (stimulate transcription)

- Silencers (inhibit transcription)

* often found nearby (-50 to -100) but can also be

found great distances away in either direction

Figure 12.11

• cis-acting elements• DNA sequences that exert their effect only on nearby

genes• Example: TATA box, enhancers and silencers

Factors that Control Gene Expression

• trans-acting elements• Regulatory proteins that bind to such DNA sequences

• In Bacteria:– DNA coding strand = mRNA sequence directly– Corresponds to amino acid sequence in the polypeptide

= colinearity of gene expression

• In Eukaryotes, RNA is modified:

- coding sequences, called exons, are interrupted by

intervening sequences or introns (which are removed)

= RNA splicing

Figure 12.16

• In eukaryotes, the transcription of structural genes, produces a long transcript known as pre-mRNA– Also as heterogeneous

nuclear RNA (hnRNA)

• RNA is altered by splicing and other modifications, before it leaves the nucleus

• Spliceosome required for splicing = multicomponent structure

• Most mature mRNAs have a 7-methyl guanosine covalently attached at their 5’ end = capping

• Occurs as pre-mRNA is being synthesized (RNA pol II)

• Cap structure is recognized by cap-binding proteins

Role of Cap-binding proteins– Movement of some RNAs into the cytoplasm– Early stages of translation– Splicing of introns

Capping

• Most mature mRNAs have a string of adenine nucleotides at their 3’ ends = polyA tail

• polyA tail is not encoded in the gene sequence– added enzymatically after the gene is completely

transcribed

Addition of poly A tail

Figure 12.20

Consensus sequence in higher eukaryotes

Appears to be important in the stability of mRNA and the

translation of the polypeptide

Length varies between species

From a few dozen adenines to several hundred

Alternative splicing:

• pre-mRNA with multiple introns can be spliced in

different ways

• Generates mature mRNAs with different

combinations of exons = different polypeptides

• Organism can carry fewer genes within genome

• Variation in splicing can occur in different cell types

or during different stages of development

Intron Advantage?