(CHAPTER 12- Brooker Text)

Feb 19, 2007 BIO 184 Dr. Tom Peavy

Sequence Complexity in the Genome

60-70% of human DNA fragments are unique DNA sequences

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

The Stages of Transcription

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

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

Initiation

• = 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

2 Prokaryotic Transcription

• E. coli RNA polymerase = holoenzyme

– Core enzyme (Four subunits = α2ββ’) – (One subunit = σ)

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

Bacterial Promoter

Sequence elements that play a key role in transcription

(pribnow box)

• 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

3 Termination of • 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 ρ (rho) –2. rho-independent termination • Does not require ρ but uses stem loop structure and U–rich sequence region

rho utilization site

Rho protein is a helicase

Figure 12.8 ρ-dependent termination

Figure 12.8 ρ-dependent termination

4 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)

TRANSCRIPTION IN

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

• 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

Figure 12.11

• 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

5 Factors that Control

• cis-acting elements • DNA sequences that exert their effect only on nearby genes • Example: TATA box, enhancers and silencers

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

• In : – 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

• 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

Figure 12.16

6 Capping • 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

Addition of poly A tail

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

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

Figure 12.20

Consensus sequence in higher eukaryotes

Appears to be important in the Length varies between species stability of mRNA and the From a few dozen translation of the polypeptide to several hundred

7 Intron Advantage?

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

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