ABSTRACT

		THE STRUCTURE AND FUNCTION OF HUMAN RAP30, 
          THE SMALL SUBUNIT OF GENERAL TRANSCRIPTION FACTOR TFIIF

				BY

			   Shi-Min Fang

	RAP30 is the small subunit of TFIIF (RAP30/74), a general initiation
and elongation factor for transcription by RNA polymerase II. RAP30 has
functions analogous to the functions of bacterial sigma factors. It binds to
RNA polymerase II, recruits RNA polymerase II to the pre-initiation complex,
prevents RNA polymerase II from binding nonspecifically to DNA, and greatly
inhibits nonspecific transcription by RNA polymerase II. Applying a
2-dimensional sequence analysis method called Hydrophobic Cluster Analysis,
we have aligned RAP30 to bacterial sigma factors. RAP30 contains sequences
weakly similar and co-linear with conserved subregions 1.2, 2.1, 3.1, and
4.1 of sigma factors. RAP30 also has sequence similar to the N-terminal
region of delta protein, a non-essential subunit of B. sublilis  RNA
polymerase. 
	In order to understand the relationship between the structure and
the function of RAP30, a set of deletion mutants of RAP30 was constructed.
Mutants were tested for accurate transcriptional activity, RAP74 binding,
RNA polymerase II binding and TFIIB binding. Transcription assays indicate
the importance of both N- and C-terminal regions for RAP30 function. RAP74
binds to the N-terminal region of RAP30 between amino acids 1-98. Two
regions of RAP30, one near the N-terminus and one within the central region,
are important for RNA polymerase II binding. Multiple contacts within the
RAP74-binding and RNA polymerase II-binding regions of RAP30 contribute to
TFIIB binding. Deletion of the N-terminal region of RAP30 abolishes
RAP74-binding, RNA polymerase II-binding and TFIIB binding and activates the
C-terminal region of RAP30 for DNA binding. The N-terminal region of RAP30,
therefore,  may be a domain that regulates the accessibility of central and
C-terminal domains.
	Several lines of evidence indicate that there may be significant
coupling of TFIIF and TFIIB function in transcription. Both RAP30 and RAP74
bind independently to TFIIB. Analysis of deletion mutants of RAP74 shows
that a C-terminal region between amino acids 358-517 binds directly to
TFIIB, and this region of RAP74 also binds to RNA polymerase II.
Interestingly, RAP74 antagonizes the interaction between TFIIB and RAP30,
both by binding to RAP30 and by binding to TFIIB. RAP30, therefore, binds to
TFIIB only in the absence of RAP74. When the TFIIF complex is intact,
TFIIF-TFIIB contact most likely is maintained through the RAP74 subunit. If
these binding relationships are maintained within functional transcription
complexes, dynamic interactions between TFIIF subunits and TFIIB may be a
mechanism to separate RAP30 and RAP74 functions during various stages of the
transcription cycle.